Peptides and combination of peptides for use in immunotherapy against small cell lung cancer and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/233,284, filed 27 Dec. 2018 (now U.S. Pat. No. 10,377,802, issued 13Aug. 2019), which is a continuation of U.S. application Ser. No.15/281,537, filed 30 Sep. 2016 (now U.S. Pat. No. 10,253,077, issued 9Apr. 2019), which claims the benefit of U.S. Provisional ApplicationSer. No. 62/237,091, filed 5 Oct. 2015, and Great Britain ApplicationNo. 1517538.3, filed 5 Oct. 2015, the content of each of theseapplications is herein incorporated by reference in their entirety.

This application also is related to PCT/EP2016/073416 filed 30 Oct.2016, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2422.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-055005_ST25.txt” createdon 6 Jan. 2020, and 22,780 bytes in size) is submitted concurrently withthe response to the Quayle Action filed on Jan. 8, 2020, and the entirecontents of the Sequence Listing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Small Cell Lung Cancer (SCLC)

Small cell lung cancer (SCLC) is named according to the size of thecancer cells when observed under a microscope and has to bedifferentiated from non-small cell lung cancer (NSCLC). SCLC accounts toabout 10% to 15% of all lung cancers (American Cancer Society, 2015a).

Both lung cancers (SCLC and NSCLC) are the second most common cancer inboth men and women. Lung cancer is leading cause of cancer death, whichaccounts for about 25%. Thus, more people die of lung cancer than ofcolon, breast, and prostate cancers combined each year. Furthermore,both lung cancers account for about 13% (more than 1.8 million) of allnew cancers. Lung cancer mainly occurs in older people. The average ageat the time of diagnosis is about 70. Fewer than 2% of all cases arediagnosed in people younger than 45. The treatment and prognosis of SCLCdepend strongly on the diagnosed cancer stage. The staging of SCLC basedon clinical results is more common than the pathologic staging. Theclinical staging uses the results of the physical examination, variousimaging tests and biopsies. According to the data introduced by AmericanCancer Society the 5-year relative survival rate accounts to 31% forstage I, 19% for stage II, 8% for stage III, and 2% for stage IV.

The standard chemo treatment of SCLC uses the combination of eitheretoposide or irinotecan with either cisplatin or carboplatin. Thetreatment is given in 4 to 6 cycles. Each cycle begins with the chemotreatment for 1 to 3 days followed by recovery period of 3 to 4 weeks.

The standard radiation therapy for treatment of SCLC is called externalbeam radiation therapy (EBRT) and usually given once or twice a day, 5days a week, for 3 to 7 weeks. In the last few years, the new radiationtechniques have been developed. The new techniques are three-dimensionalconformal radiation therapy (3D-CRT) and intensity modulated radiationtherapy (IMRT). Both of them allow the more precise targeting ofradiation load towards tumor by lowering the radiation exposure tosurrounding healthy tissue.

At the stage I, when SCLC is found as a single small tumor with noevidence of cancer spread in lymph nodes or elsewhere in general (lessthan in 1 out of 20 patients), a surgery followed by combined chemo- andradiation therapy is a standard treatment. This treatment procedure isonly an option for patients with fairly good health. Mostly, by the timewhen SCLC is diagnosed it has already spread. Thus, the treatment bysurgery is unlikely (American Cancer Society, 2015a; S3-LeitlinieLungenkarzinom, 2011).

At the limited stage, when SCLC has spread throughout one side of thechest to the lung or nearby lymph nodes (1 out of 3 patients) thecombined chemo- and radiation therapy so-called concurrentchemoradiation is a standard treatment. At this stage, the surgery isnot an option. The standard chemo drugs are etoposide (VP-16) togetherwith either cisplatin or carboplatin. The concurrent combination ofchemo—with radiation therapy showed therapeutic advantages but also isfollowed by severe side effects compared to the chemo or radiationtreatment by itself. The patients who are unlikely to tolerate theconcurrent chemoradiation, chemo therapy is a standard treatment.Optionally chemo therapy can be followed by radiation therapy (AmericanCancer Society, 2015a; S3-Leitlinie Lungenkarzinom, 2011).

At the extensive stage, when SCLC has spread widely throughout the lung,nearby lymph nodes and other distant organs (like bone marrow) thesystematic chemotherapy mostly etoposide combined with either cisplatinor carboplatin optionally followed by radiation treatment to the chestis the applied treatment (American Cancer Society, 2015a; S3-LeitlinieLungenkarzinom, 2011).

Since SCLC is known to spread to the brain, the patients with SCLCindependently on the stage will be given the prophylactic radiationtherapy to the head so-called prophylactic cranial irradiation or PCI.

At the limited and extensive stage, the treatment is likely to result insignificant shrinking of the cancer but in the most cases the cancerwill return at some point. The change in type of chemotherapy is toconsider in the cases, when cancer continues to grow in spite of appliedchemotherapy. The choice of chemotherapy by reoccurring cancer dependson the duration of cancer remission phase.

Innovations occurred regarding detection, diagnosis and treatment ofSCLC. It was shown that the usage of CT scans instead of x-rays forearly cancer detection lowered the risk of death from lung cancer.Nowadays, the diagnosis of SCLC can be supported by fluorescence orvirtual bronchoscopy, the real-time tumor imagining can be implementedby the radiation treatment. The novel anti-angiogenesis drugs likebevacizumab (Avastin), sunitinib (Sutent) and nintedanib (BIBF 1120)were shown to have therapeutic effects in treatment of SCLC (AmericanCancer Society, 2015a).

The immune therapy presents an excessively investigated field of cancertherapy. Various approaches are studded in the treatment of SCLC. One ofthe approaches targets the blocking of CTLA-4, a natural human immunesuppressor. The inhibition of CTLA-4 intends to boost the immune systemto combat the cancer. Recently, the development of promising immunecheck point inhibitors for treatment of SCLC has been started. Anotherapproach is based on anti-cancer vaccines which is currently availablefor treatment of SCLC in clinical studies (American Cancer Society,2015b; National Cancer Institute (NCI), 2011).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and small cell lung cancer in particular.There is also a need to identify factors representing biomarkers forcancer in general and small cell lung cancer in particular, leading tobetter diagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al., 2006;Dengjel et al., 2006). Elongated (longer) peptides of the invention canfunction as MHC class II active epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 126 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 126, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 126 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 126,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 are furthermore useful in thediagnosis and/or treatment of various other malignancies that involve anover-expression or over-presentation of the respective underlyingpolypeptide.

TABLE 1 Peptides according to the present invention. SEQ Official IDGene Gene No Sequence ID(s) Symbol(s)  1 AMLEEVNYI      9134 CCNE2  2VMFNFPDQATV     26960 NBEA  3 VLAEIDPKQLV     28981 IFT81  4 GLLDPGMLVNI     7182 NR2C2  5 SLQSLIISV      7398 USP1  6 SIMDYVVFV      8884SLC5A6  7 GLLGDIAIHL     84059 GPR98  8 VLIDDSQSIIFI     57380 MRS2  9AAAPGEALHTA    153572 IRX2 10 ILAAGFDGM    149175 MANEAL 11 KLFAIPILL     2328 FMO3 12 MLFEGLDLVSA     56603 CYP26B1 13 FLTAFLVQI       392ARHGAP1 14 ILIETKLVL      3708 ITPR1 15 SLLTAISEV     55086 CXorf57 16VILDLPLVI 101060503, TXNIP     10628 17 SLMLVTVEL      8546 AP3B1 18ALGEISVSV     23113 CUL9 19 VLLTTAVEV     23677 SH3BP4 20 MLDEILLQL     5425 POLD2 21 TMEEMIFEV      2140 EYA3 22 LLPEKSWEI      6898 TAT23 YQIDTVINL     50808 AK3 24 FLMEEVHMI     10057 ABCC5 25 GLSETILAV     9631 NUP155 26 KMLDEAVFQV     89796 NAV1 27 SLDIITITV     79572ATP13A3 28 ILVSQLEQL     10844 TUBGCP2 29 NLISQLTTV     22979 EFR3B 30KMLGLTVSL     51651 PTRH2 31 RLLQDPVGV      6002 RGS12 32 ALTSLELEL   163732 CITED4 33 GLYSKTSQSV     27032 ATP2C1 34 LVFEGIMEV     11215AKAP11 35 FMGDVFINV     23491 CES3 36 RMDGAVTSV      8648 NCOA1 37SLFYNELHYV    114793 FMNL2 38 GLISSLNEI      6578 SLCO2A1 39 GLDPTQFRV     5422 POLA1 40 GLLEVQVEV     84171 LOXL4 41 KAYQELLATV      8914TIMELESS 42 GLLEDERALQL     92312 MEX3A 43 YLWSEVFSM     57486 NLN 44ALIVGIPSV      7976 FZD3 45 SLSGEIILHSV    121441 NEDD1 46 ALWVAVPKA    93109 TMEM44 47 GLLEALLKI     57187 THOC2 48 SLIGLDLSSV      9765ZFYVE16 49 RLALNTPKV     23094 SIPA1L3 50 FLLSQIVAL    347051 SLC10A5 51ILDEAGVKYFL    113828 FAM83F 52 ILASFMLTGV      9931 HELZ 53 LLSEEHITL     9969 MED13 54 HLFDIILTSV     79659 DYNC2H1 55 LLIADNPQL      7404UTY 56 SLFSQMGSQYEL     79192 IRX1 57 VLIGDVLVAV     27152 INTU 58VLLNINGIDL    222484 LNX2 59 VLLSGLTEV      9498 SLC4A8 60 VVSGATETL    23547 LILRA4 61 YQAPYFLTV      9890 LPPR4 62 VMLPIGAVVMV    151258SLC38A11 63 LLMSTENEL      4602 MYB 64 VLFHQLQEI     25821 MTO1 65VMYDLITEL      3782 KCNN3 66 YLNLISTSV     55757 UGGT2 67 MLYDIVPVV   151963 MB21D2 68 FLFPVYPLI     79796, ALG9,     91893 FDXACB1 69KLFDRSVDL     55957 LIN37 70 TLLWKLVEV     54901 CDKAL1 71 FIFEQVQNV    83852 SETDB2 72 KAIGSLKEV      1894 ECT2 73 SLSSYTPDV     29843SENP1 74 FLDSLSPSV     65250 C5orf42 75 SLDLHVPSL     51750, RTEL1,     8771 TNFRSF6B 76 VLTTVMITV    166929 SGMS2

TABLE 2 Additional peptides according to thepresent invention with no prior known cancer association. SEQ OfficialID Gene Gene No Sequence ID(s) Symbol(s)  77 AIIDGKIFCV   5531 PPP4C  78RIIDPEDLKALL  29994 BAZ2B  79 RLLEPAQVQQL 152002 XXYLT1  80 ILMDPSPEYA  1786 DNMT1  81 LLAEIGAVTLV  79042 TSEN34  82 ALSSVIKEL 440145 MZT1  83KLLEIDIDGV   5422 POLA1  84 KMFENEFLL     29 ABR  85 FAYDGKDYLTL   3133HLA-E  86 KVIDYVPGI  25976 TIPARP  87 LLQNNLPAV  22948 CCT5  88TLHRETFYL   9134 CCNE2  89 IQHDLIFSL   3091 HIF1A  90 TLVDNISTMAL  55856ACOT13  91 KLQDGVHII  51118 UTP11L  92 YLQDYTDRV  10946 SF3A3  93ALRETVVEV   7415 VCP  94 ALFPVAEDISL  84164 ASCC2  95 ALYSKGILL   9631NUP155  96 NLLKLIAEV  57405 SPC25  97 ALLDGTVFEI   8214, DGCR6,  85359DGCR6L  98 ALVDHLNVGV   2647 BLOC1S1  99 QMLEAIKALEV   7690 ZNF131 100VADPETRTV  11284 PNKP 101 AMNSQILEV  23036 ZNF292 102 ALFARPDLLLL  55324ABCF3 103 SLLEYQMLV  23511 NUP188 104 TLIQFTVKL   6775 STAT4 105SMYDKVLML   9328 GTF3C5 106 KMPDDVWLV   8567 MADD 107 AMYGTKLETI   8573CASK 108 ILLDDQFQPKL  11213 IRAK3 109 SLFERLVVL   5976 UPF1 110GLTETGLYRI  29127, RACGAP1,  83956 RACGAP1P 111 FLPEAPAEL   4171 MCM2112 LLLPGVIKTV   3980 LIG3 113 LTDPDIHVL  23335 WDR7 114 ALLEPGGVLTI 23195 MDN1 115 ALLPSDCLQEA  64410 KLHL25 116 ALLVRLQEV   3695 ITGB7 117FLLDSAPLNV 134430 WDR36 118 KLPSFLANV   2585 GALK2 119 SLIDDNNEINL 65975 STK33 120 SLAADIPRL 171425 CLYBL 121 YMLEHVITL   9735 KNTC1 122SMMPDELLTSL   9994 CASP8AP2 123 KLDKNPNQV   9994 CASP8AP2 124 SLITDLQTI 26005 C2CD3 125 LLSEPSLLRTV  91442 C19orf40 126 AAASLIRLV   3064 HTT

TABLE 3 Peptides useful for e.g. personalized  cancer therapies. SEQOfficial ID Gene Gene No Sequence ID(s) Symbol(s) 127 SQAPVLDAI   1293COL6A3 128 SLAPAGVIRV  30012 TLX3 129 RVADYIVKV 201780 SLC10A4 130SLYDNQITTV   6585, SLIT1,   9353 SLIT2 131 ILMGTELTQV  10439 OLFM1 132NLLAEIHGV  10570 DPYSL4 133 IMEDIILTL   1656 DDX6 134 FMIDASVHPTL221960, CCZ1,  51622 CCZ1B 135 SLMMTIINL   7153 TOP2A 136 FLPPEHTIVYI  9896 FIG4 137 NLLELFVQL   5297 PI4KA 138 RLLDFPEAMVL  23113 CUL9 139FLSSVTYNL  23312 DMXL2 140 GLLEVMVNL  23001 WDFY3 141 NLPEYLPFV  55832,C22orf32,  91689 CAND1

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, non-small cell lungcancer, small cell lung cancer, renal cell cancer, brain cancer, gastriccancer, colorectal cancer, hepatocellular cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, ovarian cancer, urinary bladder cancer, uterine cancer,gallbladder and bile duct cancer and esophageal cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 126. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 76 (see Table 1), and their uses in theimmunotherapy of small cell lung cancer, non-small cell lung cancer,small cell lung cancer, renal cell cancer, brain cancer, gastric cancer,colorectal cancer, hepatocellular cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, urinary bladder cancer, uterine cancer, gallbladder andbile duct cancer and esophageal cancer, and preferably small cell lungcancer.

Even more preferred are the peptides—alone or in combination—selectedfrom the group consisting of SEQ ID NO: 7, 8, 33, 39, 40, 45, 47, 58,59, 73, 79, 80, 81, 88, 110, 111, 112, and 115, and their uses in theimmunotherapy of small cell lung cancer, non-small cell lung cancer,small cell lung cancer, renal cell cancer, brain cancer, gastric cancer,colorectal cancer, hepatocellular cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, urinary bladder cancer, uterine cancer, gallbladder andbile duct cancer and esophageal cancer, and preferably small cell lungcancer. Most preferred is the peptide of SEQ ID NO: 72.

As shown in the following Tables 4A and B, many of the peptidesaccording to the present invention are also found on other tumor typesand can, thus, also be used in the immunotherapy of other indications.Also refer to FIGS. 1A-1P and Example 1.

TABLE 4A Peptides according to the present invention andtheir specific uses in other proliferative di-seases than SCLC, especially in other cancerousdiseases. The table shows for selected peptideson which additional tumor types they were foundand either over-presented on more than 5% ofthe measured tumor samples, or presented onmore than 5% of the measured tumor samples witha ratio of geometric means tumor vs normal tis-sues being larger than 3. Over-presentation isdefined as higher presentation on the tumorsample as compared to the normal sample withhighest presentation. Normal tissues againstwhich over-presentation was tested were:adipose tissue, adrenal gland, blood cells,blood vessel, bone marrow, brain, esophagus,eye, gallbladder, heart, kidney, large in-testine, liver, lung, lymph node, nerve,pancreas, parathyroid gland, peritoneum,pituitary, pleura, salivary gland, skeletalmuscle, skin, small intestine, spleen,stomach, thymus, thyroid gland, trachea, ureter, urinary bladder. SEQ IDOther relevant NO: Sequence organs/diseases   1 AMLEEVNYI PC, Leukemia  2 VMFNFPDQATV PrC   3 VLAEIDPKQLV HCC, OC   4 GLLDPGMLVNIUterine Cancer   6 SIMDYVVFV GC, CRC, HCC, BRCA, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer   7 GLLGDIAIHLBrain Cancer, BRCA, Uterine  Cancer   8 VLIDDSQSIIFI Leukemia, OC   9AAAPGEALHTA NSCLC, BRCA, MCC, Esophageal  Cancer, Urinary bladder cancer 11 KLFAIPILL HCC, Gallbladder Cancer, Bile  Duct Cancer  12 MLFEGLDLVSAPrC  13 FLTAFLVQI Leukemia, Urinary bladder  cancer  14 ILIETKLVLLeukemia  15 SLLTAISEV Brain Cancer, HCC, PC, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer  16 VILDLPLVIPC, Leukemia  17 SLMLVTVEL PC, Leukemia, BRCA, Urinary  bladder cancer 18 ALGEISVSV Leukemia  19 VLLTTAVEV BRCA, Uterine Cancer  20 MLDEILLQLRCC, GC, CRC, Leukemia,  Urinary bladder cancer  21 TMEEMIFEVCRC, Leukemia, Urinary bladder  cancer, GallbladderCancer, Bile Duct Cancer  22 LLPEKSWEI HCC  23 YQIDTVINLPrC, Urinary bladder cancer  24 FLMEEVHMI NSCLC, CRC, Melanoma, Gall-bladder Cancer, Bile Duct Cancer  25 GLSETILAVNSCLC, Brain Cancer, CRC, HCC,  PC, PrC, Leukemia, BRCA,Melanoma, Esophageal Cancer, OC, Urinary bladder cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer  26 KMLDEAVFQVNSCLC, Brain Cancer, CRC,  BRCA, Melanoma, Urinary  bladder cancer  27SLDIITITV NSCLC, RCC, Brain Cancer, GC,  HCC, PC, BRCA, Melanoma, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer  28ILVSQLEQL Gallbladder Cancer, Bile Duct  Cancer  29 NLISQLTTVBrain Cancer  30 KMLGLTVSL CRC, BRCA, OC, Gallbladder Cancer, Bile Duct Cancer  31 RLLQDPVGV Brain Cancer, HCC, PrC, BRCA, Uterine Cancer  32 ALTSLELEL HCC, PrC, BRCA, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer  33 GLYSKTSQSVNSCLC, HCC, Esophageal Cancer  34 LVFEGIMEV Leukemia, Urinary bladder cancer, Uterine Cancer  37 SLFYNELHYV Melanoma  38 GLISSLNEI Leukemia 39 GLDPTQFRV Urinary bladder cancer  43 YLWSEVFSM Leukemia  45SLSGEIILHSV NSCLC, CRC, Melanoma, Urinary  bladder cancer  47 GLLEALLKILeukemia  49 RLALNTPKV HCC, Leukemia  52 ILASFMLTGV Leukemia  53LLSEEHITL Leukemia, Urinary bladder  cancer  57 VLIGDVLVAV OC  58VLLNINGIDL Urinary bladder cancer  59 VLLSGLTEV Brain Cancer, Leukemia 61 YQAPYFLTV Brain Cancer, OC, Urinary  bladder cancer, Uterine Cancer 64 VLFHQLQEI Leukemia, Melanoma  65 VMYDLITEL NSCLC, PC, BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer  66 YLNLISTSVUrinary bladder cancer,  Gallbladder Cancer, Bile Duct  Cancer  67MLYDIVPVV NSCLC, PC, OC, Urinary  bladder cancer  68 FLFPVYPLIBrain Cancer, CRC, Melanoma,  Urinary bladder cancer  69 KLFDRSVDLNSCLC, RCC, Brain Cancer, CRC,  HCC, PC, BRCA, OC, Urinarybladder cancer  72 KAIGSLKEV OC  73 SLSSYTPDV Leukemia  76 VLTTVMITVPC, OC  77 AIIDGKIFCV GC, Urinary bladder cancer  78 RIIDPEDLKALLNSCLC, CRC, HCC, Urinary  bladder cancer, Uterine Cancer  79 RLLEPAQVQQLNSCLC, Brain Cancer, HCC,  BRCA, Esophageal Cancer  80 ILMDPSPEYABrain Cancer, BRCA, MCC,  Melanoma, Urinary bladder  cancer  81LLAEIGAVTLV NSCLC, HCC, Melanoma, OC,  Urinary bladder cancer  82ALSSVIKEL CRC, Esophageal Cancer,  Urinary bladder cancer, Uterine Cancer  83 KLLEIDIDGV Brain Cancer, CRC, Leukemia, BRCA, MCC, OC, Urinary bladder cancer, Uterine Cancer  84 KMFENEFLLCRC, Leukemia, Urinary bladder  cancer, Gallbladder Cancer,Bile Duct Cancer  85 FAYDGKDYLTL RCC, Leukemia, BRCA,  Esophageal Cancer 86 KVIDYVPGI NSCLC, Brain Cancer, HCC,  BRCA, Esophageal Cancer, OC,Gallbladder Cancer, Bile Duct  Cancer  87 LLQNNLPAVLeukemia, Melanoma, OC,  Urinary bladder cancer  88 TLHRETFYLOC, Urinary bladder cancer,  Gallbladder Cancer, Bile Duct  Cancer  89IQHDLIFSL GC  90 TLVDNISTMAL CRC, OC  91 KLQDGVHIINSCLC, Gallbladder Cancer,  Bile Duct Cancer  92 YLQDYTDRV Leukemia, OC 93 ALRETVVEV NSCLC, Brain Cancer, Leukemia, BRCA, Esophageal Cancer, OC, Urinary bladder cancer,Gallbladder Cancer, Bile Duct  Cancer  94 ALFPVAEDISLNSCLC, Leukemia, Urinary  bladder cancer  95 ALYSKGILLNSCLC, RCC, CRC, HCC, MCC,  Esophageal Cancer, OC,Urinary bladder cancer  96 NLLKLIAEV RCC, Brain Cancer, CRC, HCC, Melanoma, Uterine Cancer, Gallbladder Cancer, Bile Duct  Cancer  97ALLDGTVFEI Brain Cancer, HCC, MCC, OC,  Urinary bladder cancer  98ALVDHLNVGV Brain Cancer, HCC, PrC,  Leukemia, OC, Uterine Cancer,Gallbladder Cancer, Bile Duct  Cancer  99 QMLEAIKALEVCRC, Leukemia, Melanoma,  Urinary bladder cancer 101 AMNSQILEVBrain Cancer, CRC, Leukemia,  BRCA, Uterine Cancer 102 ALFARPDLLLLLeukemia, Melanoma, OC,  Urinary bladder cancer 103 SLLEYQMLVBRCA, Urinary bladder cancer,  Gallbladder Cancer, Bile Duct  Cancer 105SMYDKVLML MCC, OC 106 KMPDDVWLV BRCA, Uterine Cancer 107 AMYGTKLETICRC, HCC, PC, OC 108 ILLDDQFQPKL Melanoma 109 SLFERLVVLHCC, MCC, Urinary bladder  cancer 110 GLTETGLYRINSCLC, CRC, PC, Leukemia,  Esophageal Cancer, OC,Gallbladder Cancer, Bile  Duct Cancer 111 FLPEAPAELLeukemia, Gallbladder Cancer,  Bile Duct Cancer 112 LLLPGVIKTV Leukemia116 ALLVRLQEV Leukemia 118 KLPSFLANV HCC, Uterine Cancer, Gallbladder Cancer, Bile  Duct Cancer 119 SLIDDNNEINL OC, Uterine Cancer121 YMLEHVITL CRC, Leukemia, MCC, Melanoma,  OC, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer 123 KLDKNPNQVLeukemia, Urinary bladder  cancer 124 SLITDLQTINSCLC, Brain Cancer, CRC, BRCA,  OC, Urinary bladder cancer,Uterine Cancer 126 AAASLIRLV Leukemia, BRCA NSCLC = non-small cell lungcancer, RCC = kidney cancer, CRC = colon or rectum cancer, GC = stomachcancer, HCC = liver cancer, PC = pancreatic cancer, PrC = prostatecancer, leukemia, BrCa = breast cancer, MCC = Merkel cell carcinoma, OC= ovarian cancer

TABLE 4B Peptides according to the present invention andtheir specific uses in other proliferative di-seases, especially in other cancerous diseases(amendment of Table 4). This table shows, likeTable 4A, for selected peptides on which ad-ditional tumor types they were found showingover-presentation (including specific presen-tation) on more than 5% of the measured tumorsamples, or presentation on more than 5% ofthe measured tumor samples with a ratio ofgeometric means tumor vs normal tissues beinglarger than 3. Over-presentation is definedas higher presentation on the tumor sample ascompared to the normal sample with highestpresentation. Normal tissues against whichover-presentation was tested were: adiposetissue, adrenal gland, artery, bone marrow,brain, central nerve, colon, duodenum,esophagus, eye, gallbladder, heart, kidney,liver, lung, lymph node, mononuclear whiteblood cells, pancreas, parathyroid gland,peripheral nerve, peritoneum, pituitary,pleura, rectum, salivary gland, skeletalmuscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, ureter, urinary bladder, vein. SEQ IDNo Sequence Additional Entities   4 GLLDPGMLVNI Brain Cancer  13FLTAFLVQI RCC  14 ILIETKLVL CLL, NHL  16 VILDLPLVI CLL, HNSCC  17SLMLVTVEL CLL, Melanoma, NHL  20 MLDEILLQL CLL, AML, NHL, HNSCC  21TMEEMIFEV CLL, Esophageal Cancer,  Uterine Cancer, HNSCC  24 FLMEEVHMIHNSCC  25 GLSETILAV CLL, AML, NHL, HNSCC  26 KMLDEAVFQV HNSCC  27SLDIITITV NHL  28 ILVSQLEQL CLL, BRCA, NHL  30 KMLGLTVSLRCC, Melanoma, AML, NHL  32 ALTSLELEL HNSCC  34 LVFEGIMEV CLL  49RLALNTPKV CLL, NHL  58 VLLNINGIDL RCC, Melanoma, Uterine Cancer, NHL, HNSCC  64 VLFHQLQEI CRC, CLL, AML, NHL  68 FLFPVYPLIUterine Cancer, AML, NHL, HNSCC  69 KLFDRSVDL Uterine Cancer, AML  72KAIGSLKEV CRC, Esophageal Cancer, Uterine  Cancer  77 AIIDGKIFCVUterine Cancer  82 ALSSVIKEL NSCLC, CLL, BRCA, Melanoma, OC, Gallbladder Cancer, Bile Duct Cancer, AML, NHL, HNSCC  83 KLLEIDIDGVMelanoma, AML, NHL, HNSCC  84 KMFENEFLL CLL, NHL, HNSCC  85 FAYDGKDYLTLCLL, Melanoma, Gallbladder  Cancer, Bile Duct Cancer, AML  86 KVIDYVPGIMelanoma, HNSCC  87 LLQNNLPAV HNSCC  89 IQHDLIFSLCLL, Melanoma, Urinary bladder  cancer, AML  91 KLQDGVHIICLL, Melanoma, AML, NHL  92 YLQDYTDRV CLL, NHL  93 ALRETVVEVMelanoma, Uterine Cancer, AML,  HNSCC  94 ALFPVAEDISLRCC, CLL, BRCA, Melanoma, AML,  NHL  95 ALYSKGILLGallbladder Cancer, Bile Duct  Cancer, NHL  96 NLLKLIAEV PC, AML, NHL 97 ALLDGTVFEI BRCA, Uterine Cancer  98 ALVDHLNVGV CLL  99 QMLEAIKALEVBrain Cancer, CLL, NHL, HNSCC 100 VADPETRTV Melanoma 101 AMNSQILEV AML102 ALFARPDLLLL CLL 105 SMYDKVLML Gallbladder Cancer, Bile Duct Cancer, AML, HNSCC 107 AMYGTKLETI RCC, Esophageal Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 108 ILLDDQFQPKL AML 109SLFERLVVL Uterine Cancer 113 LTDPDIHVL RCC, CLL, BRCA, Melanoma, AML 121YMLEHVITL NSCLC, HCC, CLL, BRCA, Uterine  Cancer, AML, NHL, HNSCC 122SMMPDELLTSL NHL 124 SLITDLQTI AML, NHL NSCLC = non-small cell lungcancer, RCC = kidney cancer, CRC = colon or rectum cancer, GC = stomachcancer, HCC = liver cancer, PC = pancreatic cancer, PrC = prostatecancer, BRCA = breast cancer, OC = ovarian cancer, NHL = non-Hodgkinlymphoma, AML = acute myeloid leukemia, CLL = chronic lymphocyticleukemia, HNSCC = head and neck squamous cell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 1, 15, 16, 17, 25, 27, 65, 67, 69, 76, 96, 107, and110 for the—in one preferred embodiment combined—treatment of pancreaticcancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 1, 8, 13, 14, 16, 17, 18, 20, 21, 25, 28, 30, 34, 38,43, 47, 49, 52, 53, 59, 64, 73, 82, 83, 84, 85, 86, 87, 89, 91, 92, 93,94, 96, 98, 99, 101, 102, 105, 108, 110, 111, 113, 112, 116, 121, 123,124, and 126 for the—in one preferred embodiment combined—treatment ofleukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 2, 12, 23, 25, 31, 32, and 98 for the—in one preferredembodiment combined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 3, 6, 11, 15, 22, 25, 27, 31, 32, 33, 49, 69, 78, 79,81, 86, 95, 96, 97, 98, 107, 109, 121, and 118 for the—in one preferredembodiment combined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 3, 8, 25 32, 57, 61, 67, 69, 72, 76, 81, 82, 83, 86,87, 88, 90, 92, 93, 95, 97, 98, 102, 105, 107, 110, 119, 121, and 124for the—in one preferred embodiment combined—treatment of ovariancancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 4, 7, 19, 21, 25, 31, 32, 34, 58, 61, 65, 68, 69, 72,77, 78, 82, 83, 93, 96, 97, 98, 101, 106, 109, 118, 119, 121, and 124for the—in one preferred embodiment combined—treatment of uterinecancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 20, 27, 77, and 89 for the—in one preferredembodiment combined—treatment of stomach cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 20, 21, 24, 25, 26, 30, 45, 64, 68, 69, 72, 78, 82,83, 84, 90, 95, 96, 99, 101, 107, 110, 121, and 124 for the—in onepreferred embodiment combined—treatment of colon or rectum cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 7, 9, 17, 19, 25, 26, 27, 28, 30, 31, 32, 65, 69,79, 80, 82, 83, 84, 85, 86, 93, 94, 101, 103, 106, 121, 124, and 126 forthe—in one preferred embodiment combined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 9, 13, 15, 17, 20, 21, 23, 25, 26, 27, 34, 39, 45,53, 58, 61, 66, 67, 68, 69, 77, 78, 80, 81, 82, 83, 84, 87, 88, 89, 93,94, 95, 97, 99, 102, 103, 109, 121, 123, and 124 for the—in onepreferred embodiment combined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 11, 15, 21, 24, 25, 27, 28, 30, 32, 65, 66, 84, 85,86, 87, 88, 91, 92, 93, 95, 96, 97, 98, 103, 105, 107, 110, 111, 118,and 121 for the—in one preferred embodiment combined—treatment ofgallbladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 6, 11, 15, 21, 24, 25, 27, 28, 30, 32, 65, 66, 84, 85,86, 87, 88, 91, 92, 93, 95, 96, 97, 98, 103, 105, 107, 110, 111, 118,and 121 for the—in one preferred embodiment combined—treatment of bileduct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 4, 7, 15, 25, 26, 27, 29, 30, 31, 59, 61, 68, 69, 79,80, 83, 86, 93, 96, 97, 98, 101, and 124 for the—in one preferredembodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 9, 24, 25, 26, 27, 33, 45, 65, 67, 69, 78, 79, 81, 82,86, 91, 93, 94, 95, 110, 121, and 124 for the—in one preferredembodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 9, 80, 83, 95, 97, 105, 109, and 121 for the—in onepreferred embodiment combined—treatment of Merkel cell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 9, 21, 25, 33, 72, 79, 82, 85, 86, 93, 95, 107, and110 for the—in one preferred embodiment combined—treatment of esophagealcancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 17, 24, 25, 26, 27, 30, 37, 45, 58, 64, 68, 80, 81,82, 83, 85, 86, 87, 89, 91, 93, 94, 96, 99, 100, 101, 102, 108, 113, and121 for the—in one preferred embodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 16, 20, 21, 24, 25, 26, 32, 58, 68, 82, 83, 84, 86,87, 93, 99, 105, and 121 for the—in one preferred embodimentcombined—treatment of head and neck squamous cell carcinoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID NO: 14, 17, 20, 25, 27, 28, 30, 49, 58, 64, 68, 82, 83,84, 91, 92, 94, 95, 96, 99, 107, 121, 122, and 124 for the—in onepreferred embodiment combined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofsmall cell lung cancer, non-small cell lung cancer, small cell lungcancer, renal cell cancer, brain cancer, gastric cancer, colorectalcancer, hepatocellular cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovariancancer, urinary bladder cancer, uterine cancer, gallbladder and bileduct cancer and esophageal cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 126.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID NO: 1 to SEQ ID NO: 126, preferably containing SEQ IDNO: 1 to SEQ ID NO: 76, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are small cell lung cancer,non-small cell lung cancer, small cell lung cancer, renal cell cancer,brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer and esophageal cancer, andpreferably small cell lung cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”,that can be used in the diagnosis of cancer, preferably small cell lungcancer. The marker can be over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment. Both therapeutic and diagnostic usesagainst additional cancerous diseases are disclosed in the followingmore detailed description of the underlying expression products(polypeptides) of the peptides according to the invention.

ABCC5 is up-regulated in different cancer entities for examplepancreatic cancer, in breast cancer metastasis compared to primarybreast cancer and in esophageal cancer with an amplified region ofchromosome 3q. ABCC5 is further frequently mutated in microsatelliteinstable (MSI) colorectal cancer (Mohelnikova-Duchonova et al., 2013;Chen et al., 2008; Alhopuro et al., 2012; Mourskaia et al., 2012). ABCC5expression is influenced by the estrogen metabolism as well as elevatedlevels of HES1, DELTEX1 and c-Myc (Larson et al., 2009; Vendrell et al.,2004).

ABCF3 gene locus shows frequent chromosomal gains correlating withcervical cancer. Moreover, ABCF3 enhances proliferation of human livercancer cells (Choi et al., 2007; Zhou et al., 2013b). ABCF3 binds to thetumor protein D52 family member TPD52L2 and positively regulates cellproliferation (Zhou et al., 2013b).

Deletion mapping of medulloblastoma tumors reveals loss of distalchromosome 17p13.3 sequences also in the ABR gene (McDonald et al.,1994).

AK3 is down-regulated in hepatocellular carcinomas and over-expressed inB-cell chronic lymphocytic leukemia (Carlucci et al., 2009; Melle etal., 2007).

AKAP11 is frequently altered and significantly over-expressed in oraltumors and further associated with cancer progression (Garnis et al.,2005). AKAP11 promotes cell migration in human cancer cells viasuppression of GSK-3beta and interaction with cytoskeletal scaffoldingproteins. AKAP11 contributes to alterations in cell cycle regulation byinfluencing the Rb pathway (Logue et al., 2011; Garnis et al., 2005).

The expression of ALG9 during TGFbeta-induced epithelial-to-mesenchymaltransition (EMT) is significantly changed and influences the N-glycanprofile of cancer cells (Tan et al., 2014).

AP3B1 is down-regulated in cervical tumors in comparison with normaltissue of the uterine cervix (Petrenko et al., 2006). AP3B1 is a targetof the microRNA miR-9, which is de-regulated in many cancer typesincluding hepatocellular and breast cancer (Zhang et al., 2015a;Selcuklu et al., 2012).

The expression of ARHGAP1 is altered in different cancer types includingprostate and metastatic brain cancer (Davalieva et al., 2015; Zohrabianet al., 2007). ARHGAP1 down-regulation by miR-34a repressesTGF-beta-induced tumor cell invasion. ARHGAP1 is associated withepithelial-to-mesenchymal transition (EMT) by restricting Rho activationwhich is necessary for detachment (Ahn et al., 2012; Clay and Halloran,2013).

ATP13A3 expression is altered in cervical cancer (Bierkens et al.,2013).

ATP2C1 is over-expressed in cervical cancer via a common chromosomalgain of the locus. Loss of ATP2C1 in mice causes increased apoptosis anda genetic predisposition to squamous cell carcinomas of the skin and theesophagus in adult heterozygotes (Wilting et al., 2008; Okunade et al.,2007). ATP2C1 inhibition produces a pronounced alteration in theprocessing of the protein IGF1R, which is important for tumorprogression (Grice et al., 2010).

BLOC1S1 is down-regulated in malignant prostate in comparison to normalprostate tissue (Asmann et al., 2002). BLOC1S1 is important for a properEGFR lysosomal trafficking also via the interaction to its partners SNX2and TSG101 (Zhang et al., 2014). C19orf40 (also called FAAP24) encodes acomponent of the Fanconi anemia (FA) core complex which plays a crucialrole in DNA damage response (RefSeq, 2002). C19orf40 builds togetherwith FANCM a complex important for DNA damage recognition, suppressionof sister chromatid exchange as well as ATR-mediated checkpointactivation in DNA damage repair and replication to ensure chromosomalstability. Alterations of C19orf40 are associated with cancer-proneFanconi anemia (Valeri et al., 2011; Wang et al., 2013d; Ciccia et al.,2007).

C2CD3 was shown to be associated with oropharyngeal squamous cellcarcinomas (Wang et al., 2013b).

CAND1 is associated with prostate cancer and lung cancer (Zhai et al.,2014; Salon et al., 2007).

CASK is over-expressed in different cancer types including gastric andcolorectal cancer as well as leukemia. Moreover, CASK is associated withcancer progression and poor prognosis (Wei et al., 2014; Zhou et al.,2014c; Al-Lamki et al., 2005). CASK is up-regulated via a PKA-dependentpathway by exendin-4 during the stimulation of beta-cell insulinsecretion and by NecI-2 together with E-cadherin during wound healingprocesses (Giangreco et al., 2009; Zhu et al., 2014).

CASP8AP2 is de-regulated in different cancer types includingdown-regulation and hyper-methylation in acute lymphoblastic leukemia,hypo-methylation in early-stage liver cancer and frequent mutations inmismatch repair-deficient colorectal cancer (Park et al., 2002; Chen etal., 2012; Li et al., 2013; Juarez-Velazquez et al., 2014). CASP8AP2 isinvolved in the regulation of targets of the transcription factorsNF-kappaB, c-Myb and Myc. Moreover, loss of function of CASP8AP2 inducethe expression of the tumor suppressor gene NEFH (Hummon et al., 2012;Alm-Kristiansen et al., 2008).

CCNE2 is over-expressed in different cancer types including breast,lung, pancreatic and bladder cancer and can further be used asprognostic marker (Deng et al., 2013; Payton et al., 2002; Gudas et al.,1999; Chen et al., 2015a; Matsushita et al., 2015; Sieuwerts et al.,2006). CCNE2 is regulated by different factors including estrogen, PTENand microRNAs. Moreover, elevated levels of CCNE2 lead to genomicinstability with abnormal mitosis, micronuclei and chromosomalaberrations (Wu et al., 2009; Caldon et al., 2009; Caldon et al., 2013;Chen et al., 2015a).

CCT5 is associated with breast cancer (Campone et al., 2008). CCT5 wasshown to be up-regulated in sinonasal adenocarcinoma (Tripodi et al.,2009). CCT5 is associated with overall survival in small cell lungcancer, drug resistance in gastric carcinoma and breast cancer and lymphnode metastasis in esophageal squamous cell carcinoma (Niu et al., 2012;Ooe et al., 2007; Uchikado et al., 2006; Ludwig et al., 2002).

CDKAL1 is frequently amplified and over-expressed in bladder cancer andsingle nucleotide polymorphisms of CDKAL1 are associated with cancerrisk, for example for colorectal cancer in men, in patients withdiabetes (Sainz et al., 2012; Ma et al., 2014b; Hurst et al., 2008).

CES3 is often up-regulated in human colon tumor tissue with largeinterindividual variations (Sanghani et al., 2003).

CITED4 is down-regulated by promoter hyper-methylation in differentcancer types including breast cancer and oligodendroglial tumors whereit is associated with prognosis (Huang et al., 2011; Tews et al., 2007).CITED4 blocks the binding of hypoxia-inducible factor 1 alpha(HIF1alpha) to p300 and inhibits HIF1 alpha transactivation andhypoxia-mediated gene activation (Huang et al., 2011; Fox et al., 2004).

COL6A3 encodes the alpha-3 chain of type VI collagen, a beaded filamentcollagen found in most connective tissues, playing an important role inthe organization of matrix components (RefSeq, 2002). COL6A3 isalternatively spliced in colon, bladder and prostate cancer. The longisoform of COL6A3 is expressed almost exclusively in cancer samples andcould potentially serve as a new cancer marker (Thorsen et al., 2008).COL6A3 is highly expressed in pancreatic ductal adenocarcinoma tissueand undergoes tumor-specific alternative splicing (Kang et al., 2014).COL6A3 has been demonstrated to correlate with high-grade ovarian cancerand contributes to cisplatin resistance. COL6A3 was observed to befrequently over-expressed in gastric cancer tissues (Xie et al., 2014).COL6A3 mutation(s) significantly predicted a better overall survival inpatients with colorectal carcinoma independent of tumor differentiationand TNM staging (Yu et al., 2015b). COL6A3 expression was reported to beincreased in pancreatic cancer, colon cancer, gastric cancer,mucoepidermoid carcinomas and ovarian cancer. Cancer associatedtranscript variants including exons 3, 4 and 6 were detected in coloncancer, bladder cancer, prostate cancer and pancreatic cancer (Arafat etal., 2011; Smith et al., 2009; Yang et al., 2007; Xie et al., 2014;Leivo et al., 2005; Sherman-Baust et al., 2003; Gardina et al., 2006;Thorsen et al., 2008). In ovarian cancer COL6A3 levels correlated withhigher tumor grade and in pancreatic cancer COL6A3 was shown torepresent a suitable diagnostic serum biomarker (Sherman-Baust et al.,2003; Kang et al., 2014).

CUL9-mediated degradation of cytochrome c is a strategy of cancer cellsto prevent apoptosis during mitochondrial stress (Gama et al., 2014).The tumor suppressor CUL9 binds to p53 and promotes p53-mediatedapoptosis. CUL9 also is a critical regulator in controlling thesubcellular localization of p53 which is essential for its function.Depletion of CUL9 results in spontaneous tumor development (Pei et al.,2011; Nikolaev et al., 2003).

CXorf57 is a common viral integration site in ALV-induced B-celllymphomas leading to a disruption of the normal gene transcription,suggesting that it could be a novel tumor suppressor (Justice et al.,2015).

Hypermethylation of CYP26B1 is a potential diagnosis marker in gastriccancer and CYB26B1 expression increases with malignancy in gliomas(Campos et al., 2011; Zheng et al., 2011). Clearance of the activemetabolite all-trans-retinoic acid (atRA) by CYP26B1 influencesregulation of differentiation, growth and migration of immune cells andis inhibited by TGF-beta, yet enhanced by TNF-alpha (Stevison et al.,2015).

DDX6 was found to be over-expressed in colorectal adenocarcinomas,gastric cancer, hepatocellular carcinoma, nodal marginal zone lymphoma,neuroblastoma, rhabdomyosarcoma and lung cancer cell lines (Akao et al.,1995; Nakagawa et al., 1999; Miyaji et al., 2003; Lin et al., 2008a;Stary et al., 2013; Iio et al., 2013). In nodal marginal zone lymphoma,DDX6 seems to interfere with the expression of BCL6 and BCL2 in an NF-κBindependent manner (Stary et al., 2013). Recent studies have shown thatDDX6 post-transcriptionally down-regulated miR-143/145 expression byprompting the degradation of its host gene product, NCR143/145 RNA (Iioet al., 2013).

DGCR6 is de-regulated in metastatic breast cancer cells and a possiblemediator of cell invasion (Euer et al., 2002).

DGCR6L regulates the migration of human gastric cancer cells via theformation of a PAK/DGCR6L/beta-actin complex (Li et al., 2010b).

DMXL2 was shown to be up-regulated in ER-alpha positive breast cancer(Faronato et al., 2015). DMXL2 is a functional biomarker for ER-alphapositive breast cancer (Faronato et al., 2015).

DNMT1 is associated with DNA methylation changes in cancer. Furthermore,DNMT1 is over-expressed in different cancer entities includingcolorectal, lung, pancreatic, prostate and liver cancer (Xu et al.,2010; He et al., 2011; Feng et al., 2014; Samaei et al., 2014;Bashtrykov and Jeltsch, 2015; Zhang et al., 2015d; Saito et al., 2003).DNMT1 stability is regulated via the Wnt-pathway, the II-6/JAK2/STAT3signaling and pRB protein.

Moreover, DNMT1 activity leads to epigenetic silencing of several tumorsuppressor genes including p16INK, p53 and p21 by promoterhyper-methylation (Rhee et al., 2002; Shamma et al., 2013; Liu et al.,2015a; Song et al., 2015).

The DPYSL4 gene is localized to chromosome 10q25.2-q26 a regionfrequently mutated in glioblastomas and which contains many tumorsuppressor genes (Honnorat et al., 1999). DPYSL4 is anapoptosis-inducible factor directly controlled by the tumor suppressorp53 in response to DNA damage (Kimura et al., 2011).

DYNC2H1 was shown to be up-regulated in glioblastoma multiforme (Yokotaet al., 2006).

ECT2 is over-expressed as a result of tumor-specific gene amplificationsin a variety of human tumors including lung, ovarian, gastric andpancreatic cancer. ECT2 is important for cell proliferation, migration,invasion and tumorigenicity (Fields and Justilien, 2010; Jin et al.,2014). Protein kinase C iota and ECT2 activate through MEK/ERK signalinga tumor-initiating cell phenotype in ovarian cancer (Wang et al.,2013c). Nuclear ECT2 is binding preferentially to the Rho GTPase Rac1and leads through Rac1 activation to cellular transformation, whilecytoplasmic ECT2 binds to the Rho GTPase RhoA and leads through RhoAactivation to the formation of cytokinetic furrow (Su et al., 2011; Huffet al., 2013).

The increased expression of EFR3B is associated with the progression ofsquamous dysplasia of the esophageal mucosa (Joshi et al., 2006). Theloss of EFR3B constitutes a rare copy number variation (CNV) detected inhereditary nonpolyposis colorectal cancer (Lynch syndrome) (Villacis etal., 2016).

EYA3 is highly expressed in Ewing sarcoma tumor samples and cell linescompared with mesenchymal stem cells. On the other hand, deletion of theEYA3 gene has been linked to certain pancreatic ductal adenocarcinomas(Gutierrez et al., 2011; Robin et al., 2012). Recent work has shown thatover-expression of EYA3 results in increased proliferation, migration,invasion and transformation of breast cancer cells (Pandey et al.,2010).

FAM83F is up-regulated in esophageal squamous cell carcinomas. Moreover,FAM83F is a target of miR-143 which inhibits proliferation, migrationand invasion. FAM83F contains as part of the family with sequencesimilarity 83 a highly conserved domain associated with driving cellulartransformation (Cipriano et al., 2014; Mao et al., 2016).

Over-expression of FIG. 4 was found in the triple negative breast cancercompared to non-tumorigenic cells (lkonomov et al., 2013).

FMNL2 is over-expressed in colorectal cancer, associated with invasionand migration and a target of different microRNAs (Zhu et al., 2008; Luet al., 2015; Ren et al., 2016). FMNL2 activates the Rho/ROCK pathway,SRF transcription and actin assembly influencing cell invasion.Moreover, FMNL2 is involved in TGF-beta-inducedepithelial-to-mesenchymal-transition and cell invasion via Smad3 andMAPK/MEK signaling. Furthermore, FMNL2 drives beta1-integrininternalization and thereby increases invasive motility (Li et al.,2010c; Wang et al., 2015c; Zeng et al., 2015).

FMO3 is differentially expressed when comparing tobacco-exposed lungtissue of smokers with non-smokers and with adenocarcinomas from smokersand may therefore be utilized to identify smokers with increased riskfor lung cancer (Woenckhaus et al., 2006). FMO3 influences the efficacyand toxicity of different cancer drugs including daunorubicin, imatiniband sulindac (Thompson et al., 2014; Hisamuddin et al., 2005; Rochat etal., 2008). FMO3 activity can be influenced by nitric oxide donors andthe recruitment of p53 to a p53 response element in the 5′-flankingregion of the gene (Celius et al., 2010; Ryu et al., 2004).

FZD3 is up-regulated in different cancer types including colorectal,gastric and liver cancer as well as leukemia and is further associatedwith cancer progression (Wong et al., 2013; Lu et al., 2004; Bengocheaet al., 2008). Binding of the ligand Wnt5a by the FZD3 receptor promotesthe activation of PI3K and Akt (Kawasaki et al., 2007).

GALK2 was identified in an in-tumor genetic screen as a potentialtherapeutic target in ovarian carcinoma (Baratta et al., 2015). GALK2 isa kinase that is involved in the regulation of prostate cancer cellgrowth identified in an RNAi phenotypic screening (Whitworth et al.,2012).

GPR98 expression is increased in primary neuroendocrine tumors relativeto normal tissue (Sherman et al., 2013). GPR98 was among the genesassociated with survival of glioblastoma multiforme (Sadeque et al.,2012). GPR98 displays a transcript regulated by glucocorticoids whichare used for the treatment of acute lymphoblastic leukemia as they leadto the induction of apoptosis (Rainer et al., 2012).

GTF3C5 is over-expressed in human ovarian carcinomas (Winter et al.,2000).

HELZ is down-regulated in different human cancer cells and located in afrequently lost chromosomal band in various human cancers. Moreover,HELZ plays a role in cancer cell growth (Nagai et al., 2003). HELZ isrepressed by beta-catenin/TCF4 activity via different microRNAs incolorectal cancer cells. HELZ interacts directly with SMYD3, which isitself frequently over-expressed in human cancers, to activatetranscription of oncogenes and is thereby contributing to carcinogenesis(Schepeler et al., 2012; Hamamoto et al., 2004).

HIF1A was shown to be associated with tumor necrosis in aggressiveendometrial cancer. HIF1A was further described to be a potential targetfor the treatment of this disease (Bredholt et al., 2015). HIF1A wasshown to be associated with hepatocarcinogenesis, sarcoma metastasis andnasopharyngeal carcinoma (Chen et al., 2014; El-Naggar et al., 2015; Liet al., 2015c). A single nucleotide polymorphism in HIF1A was shown tobe significantly associated with clinical outcomes of aggressivehepatocellular carcinoma patients after surgery (Guo et al., 2015).Aberrant HIF1A activity together with aberrant STAT3 activity was shownto drive tumor progression in malignant peripheral nerve sheath tumorcell lines. Thus, inhibition of the STAT3/HIF1A/VEGF-A signaling axiswas described as a viable treatment strategy (Rad et al., 2015). HIF1Awas described as an important target for hypoxia-driven drug resistancein multiple myeloma (Maiso et al., 2015). HIF1A was shown to beasymmetrical expressed in three different cell lines that correspondwith the stages of multiple myeloma pathogenesis, suggesting that HIF1Ais involved in the tumorigenesis and metastasis of multiple myeloma(Zhao et al., 2014a). The long noncoding HIF1A antisense RNA-2 wasdescribed as being up-regulated in non-papillary clear-cell renalcarcinomas and gastric cancer and is associated with tumor cellproliferation and poor prognosis in gastric cancer (Chen et al., 2015d).De-regulation of the PI3K/AKT/mTOR pathway through HIF1A was describedto be critical for quiescence, maintenance and survival of prostatecancer stem cells (Marhold et al., 2015). HIF1A was described as onegene of a 4-gene classifier which is prognostic for stage I lungadenocarcinoma (Okayama et al., 2014). A polymorphism of HIF1A was shownto be associated with increased susceptibility to digestive tract cancerin Asian populations (Xu et al., 2014b). HIF1A was described as aprognostic marker in sporadic male breast cancer (Deb et al., 2014).

HLA-E is over-expressed in different cancer entities including gastric,colorectal, lung and skin cancer and overexpression is associated withpoor prognosis (Allard et al., 2011; Bossard et al., 2012; Ishigami etal., 2015; Talebian et al., 2016). HLA-E/NKG2A mediates resistance oftumor cells to natural killer (NK) cell mediated lysis (Enqvist et al.,2011; He et al., 2014; Talebian et al., 2016).

HTT is associated with cancer prognosis in different cancer typesincluding breast and ovarian cancer at which wild-type HTT isdown-regulated in metastatic breast cancer. Mutant HTT leads to cancerprogression and metastasis and also the CAG repeat size seems to play arole for cancer prognosis (Moreira et al., 2013; Thion et al., 2015;Thion et al., 2016). p53 regulates the expression of HTT and the mutantHTT form modifies p53 and the p53-mediated signaling cascade leading toa slow accumulation of DNA damage (Illuzzi et al., 2011; Feng et al.,2006).

Down-regulation, promoter methylation and/or SNPs of IRAK3 in differentcancer types including liver, prostate, pancreatic, breast and brain areassociated with tumor progression and poor prognosis (Lee et al., 2009;Rajaraman et al., 2009; Caba et al., 2014; Jain et al., 2014; Kuo etal., 2015; Angulo et al., 2016). On one hand TGF-beta induces the IRAK3expression in tumor-associated macrophages (TAMs) to circumvent theanti-tumor responses of macrophages by secreting IL-12, TNFalpha andIFN-gamma. On the other IRAK3 mediates a TLR7-induced MEKK3-dependentsecond wave of NF-kappaB activation to produce an immunosuppressivefeedback (Standiford et al., 2011; Zhou et al., 2013a; Jain et al.,2014; del et al., 2005).

IRX1 is hyper-methylated and down-regulated in different cancer typesincluding gastric, oral, head and neck and bladder cancer. It is furtherassociated with cell growth and invasion (Marcinkiewicz and Gudas, 2014;Kitchen et al., 2016; Guo et al., 2010; Bennett et al., 2008b). IRX1influences metastasis formation via BDKRB2/PAK1 and is involved in theTGFbeta signaling pathway (Jiang et al., 2011; Bennett et al., 2008b).

IRX2 is de-regulated in different cancer types including over-expressionand gene amplification in sarcoma and breast cancer. On the other hand,hyper-methylation in lung cancer and loss of heterozygosity (LOH) ingastric cancer were further observed (Rauch et al., 2012; Kadota et al.,2009; Liu et al., 2014d; Yu et al., 2006; Adamowicz et al., 2006). IRX2promotes the activation of the PI3K/Akt pathway as well as increasedproliferation and invasion following the up-regulation of VEGF, MMP2 andMMP9 (Liu et al., 2014d; Liu et al., 2015c).

ITGB7 is over-expressed in different cancer types including leukemia,colorectal cancer and hepatoma cancer cells (Ortega et al., 2010; Richeset al., 2014; Liu et al., 2003; Chen et al., 1999). The oncogene c-Mafup-regulates ITGB7 and cyclin D2 leading to a malignant transformationof T-cell lymphoma cells (Morito et al., 2006).

It has been reported that the expression of ITPR1 is altered intamoxifen resistance breast cancer cell lines (Elias et al., 2015).Researchers have postulated a role for the HIF2alpha/ITPR1 axis inregulating clear cell renal cell carcinomas cell survival. In addition,ITPR1 was significantly correlated with overall survival in breastcancer (Messai et al., 2014; Gu et al., 2016).

An unusual KCNN3 expression leads to plasma membrane hyperpolarizationand enhanced cell motility of melanoma cells (Chantome et al., 2009).KCNN3 (also called TASK-1) expression was down-regulated by17beta-estradiol in mouse neuroblastoma N2A cells and improved cellproliferation (Hao et al., 2014). KCNN3 expression was up-regulated byexposure of breast cancer organotypic culture to 1,25 dihydroxy vitaminD(3) in physiological and supra-physiological concentrations (Milani etal., 2013). KCNN3 (also called K2P3.1) together with K2P1.1 and K2P12.1,were over-expressed in a range of cancers examined using the onlinecancer microarray database, Oncomine (www.oncomine.org) (Williams etal., 2013).

KNTC1 is down-regulated in oral squamous cell carcinomas with highertumor size correlating with poor prognosis. Furthermore, Frameshiftmutations of KNTC1 were detected in gastric and colorectal cancer withmicrosatellite instability (Wang et al., 2004; Diniz et al., 2015; Kimet al., 2010).

Single nucleotide polymorphisms in the LIG3 gene modify the risk fordifferent cancer types including lung, colorectal, esophageal andpancreatic cancer (Li et al., 2009; Corral et al., 2013; Hao et al.,2004; Landi et al., 2006). c-Myc plays a role in transcriptionalactivation of LIG3, leading to an increase in error-prone repair inleukemia (Muvarak et al., 2015).

A single nucleotide polymorphism in LILRA4 influences the prognosis ofpatients with chronic lymphocytic leukemia with regard to their responseto chemotherapy and overall survival (Sellick et al., 2008). PDCsexpressing LILRA4 can activate an immune-receptor tyrosine-basedactivation motif (ITAM)-mediated signaling pathway and interact withbone marrow stromal cell antigen 2 to assure an appropriate TLRresponse.by PDCs during viral infections and likely participates in PDCtumor crosstalk. Immunoglobulin-like transcript 7 ligands inhibit PDCsproduction of type I IFNs and ILT7/ILT7L interaction may thereforepresent a negative feedback following viral infection and a mechanismfor the impairment of PDCs in the cancer microenvironment (Tsukamoto etal., 2009; Palma et al., 2012).

LNX2 is de-regulated in different cancer types including a detection ofa genetic variant in diffuse large B-cell lymphoma and geneamplification with over-expression in colorectal carcinomas (Kumar etal., 2011; Camps et al., 2013). LNX2 increases Notch levels andup-regulates the transcription factor TCF7L2 and thereby activates Wntsignaling (Camps et al., 2013).

LOXL4 is de-regulated in different cancer types includingdown-regulation in liver cancer associated with poor prognosis andhyper-methylation followed by down-regulation in bladder cancer. On theother hand, over-expression in colorectal and head and neck cancer aswell as up-regulation in gastric cancer associated with tumorprogression, metastasis and poor prognosis were observed (Kim et al.,2009; Li et al., 2015a; Tian et al., 2015; Gorogh et al., 2016; Wu etal., 2007). TGF-betal induces LOXL4 following inhibition of the Ras/ERKsignaling, suppression of MMP2 activity and activation of the FAK/Srcpathway (Li et al., 2015a; Wu et al., 2007; Kim et al., 2008).

LPPR4 shows an aberrant DNA methylation pattern in pediatric B-cellacute lymphoblastic leukemia (Wong et al., 2012; Figueroa et al., 2013).

Over-expression of MADD has been found in many types of human tumors,including non-small cell lung cancer, lung adenocarcinoma, squamous cellcarcinoma, thyroid cancer, breast cancer and ovarian cancer (Subramanianet al., 2009; Li et al., 2011a; Wei et al., 2012; Bi et al., 2013;Turner et al., 2013). Researchers have demonstrated that elevated levelsof MADD in the A549 cells inhibited apoptosis and increased survival,while knock-down of MADD promoted apoptosis and reduced cellproliferation (Wei et al., 2012; Bi et al., 2013). Additionally, MADDfunction is regulated by PTEN-PI3K-Akt signaling pathway (Jayarama etal., 2014).

MANEAL is a cancer-related gene with aberrant methylation for example inbreast cancer cells, dependent on their estrogen and progesterone status(Li et al., 2010a; Liu et al., 2011).

MB21 D2 is mutated in small cell lung cancer (Peifer et al., 2012).

MCM2 has been shown to be the most sensitive marker of proliferation andprognosis in early breast cancer, renal cell carcinomas, esophageal andlaryngeal squamous cell carcinoma and oligodendroglioma of the brain(Wharton et al., 2001; Going et al., 2002; Rodins et al., 2002; Gonzalezet al., 2003; Cai et al., 2012; Joshi et al., 2015).

MDN1 was described to be a candidate tumor suppressor gene, mutated inbreast cancers of the luminal B type (Cornen et al., 2014).

MED13 is frequently amplified in breast cancer where an over-expressionis associated with poor prognosis. Moreover, frameshift mutations inMED13 are described for colorectal and gastric cancer withmicrosatellite instability (Monni et al., 2001; Broude et al., 2015; Joet al., 2016). MED13 physically links the CDK8 oncogene to the mediatorcomplex and thereby influences the function in signal-dependent generegulation (Davis et al., 2013; Clark et al., 2015).

MEX3A is over-expressed and the gene is amplified in Wilms tumorsassociated with a late relapse (Krepischi et al., 2016). MEX3A regulatesCDX2 via a post-transcriptional mechanism with impact in intestinaldifferentiation, polarity and stemness, contributing to intestinalhomeostasis and carcinogenesis (Pereira et al., 2013).

MRS2 promotes cell growth, down-regulates p27 and up-regulates cyclin D1in multidrug-resistant gastric cancer cells (Chen et al., 2009).

MTO1 is up-regulated in breast cancer cells and the expression levelinversely correlates with promoter methylation status (Kim et al.,2013).

MYB can be converted into an oncogenic transforming protein through afew mutations (Zhou and Ness, 2011). MYB is known as oncogene and isassociated with apoptosis, cell cycle control, cell growth/angiogenesisand cell adhesion by regulating expression of key target genes such ascyclooxygenase-2, Bcl-2, BcIX(L) and c-Myc (Ramsay et al., 2003; Stenmanet al., 2010). The oncogenic fusion protein MYB-NFIB and MYBover-expression are found in adenoid cystic carcinoma of the salivarygland and breast, pediatric diffuse gliomas, acute myeloid leukemia andpancreatic cancer (Wallrapp et al., 1999; Pattabiraman and Gonda, 2013;Nobusawa et al., 2014; Chae et al., 2015; Marchio et al., 2010). By thesynergy between MYB and beta-Catenin during Wnt signaling, MYB isassociated with colon tumorigenesis (Burgess et al., 2011). Since MYB isa direct target of estrogen signaling anti-MYB therapy is considered forER-positive breast tumors (Gonda et al., 2008).

The MZT1 gene is located in a chromosomal region with a putative breastcancer susceptibility gene and a common site for somatic deletions in avariety of malignant tumors (Rozenblum et al., 2002).

NAV1 is significantly hypomethylated in ER+/PR+ breast cancers andfrequently mutated in neuroblastoma (Li et al., 2010a; Lasorsa et al.,2016).

NBEA is one of the common fragile site genes inactivated in differentcancers including myelomas and oropharyngeal squamous cell carcinomas.NBEA shows frequent mutations with prognostic relevance in gastriccancer (Li et al., 2016; O'Neal et al., 2009; Nagoshi et al., 2012; Gaoet al., 2014; McAvoy et al., 2007).

NCOA1 is frequently overexpressed and further associated with cancerprogression and prognosis in different cancer types including breast,prostate and head and neck cancer (Qin et al., 2011; Qin et al., 2014;Pavon et al., 2016; Luef et al., 2016). NCOA1 promotes breast cancermetastasis by enhancing CSF1 promoter activity via an AP1 binding siteand upregulation of ITGA5 expression (Qin et al., 2011; Qin et al.,2014).

NR2C2 is associated with invasion and metastasis in different cancertypes including prostate and lung cancer (Ding et al., 2015; Qiu et al.,2015; Zhang et al., 2015b). NR2C2 influences carcinogenesis viacross-talk with other nuclear receptor pathways like PPARgamma and RARand increases invasion and metastasis through activation of theTGFbetaR2/p-Smad3 signaling (Liu et al., 2014c; Qiu et al., 2015; Lee etal., 2004).

NUP155 was described as a potential epigenetic biomarker of white bloodcell's DNA which is associated with breast cancer predisposition(Khakpour et al., 2015). NUP155 was described as strictly required forthe proliferation and survival of NUP214-ABL1-positive T-cell acutelymphoblastic leukemia cells and thus constitutes a potential drugtarget in this disease (De et al., 2014).

siRNAs targeting MAPK inhibit cervical cancer cell line growth and leadto a down-regulation of NUP188 (Huang et al., 2008; Yuan et al., 2010).NUP188 seems to be a target of the tumor suppressor gene BRCA1 in breastcancer (Bennett et al., 2008a). NUP188 is required for the chromosomealignment in mitosis through K-fiber formation and recruitment of NUMAto the spindle poles (Itoh et al., 2013).

OLFM1 is deregulated in different cancer types including endometrialcancer and lung adenocarcinomas (Wu et al., 2010; Wong et al., 2007).Up-regulation of the OLFM1 gene suppresses the activities ofextracellular inhibitors of the Wnt signaling pathway and this maypromote cell proliferation (Tong et al., 2014).

Elevated levels of PI4KA were observed in hepatocellular carcinomaversus normal liver tissue. In addition, the PI4KA gene was detected inpancreatic cancer cell line (Ishikawa et al., 2003; Ilboudo et al.,2014). Patients suffering from hepatocellular carcinoma with higherPI4KA mRNA concentrations had a higher risk of tumor recurrence as wellas shorter disease-specific survival (Ilboudo et al., 2014). Recently,PI4KA has been identified to be involved in cell proliferation andresistance to cisplatin treatment in a medulloblastoma cell line. Othershave revealed that PI4KA plays a crucial role in invasion and metastasisin pancreatic cancer (Ishikawa et al., 2003; Guerreiro et al., 2011).

POLA1 is up-regulated in tumors and high-grade intraepithelial lesionsof the uterine cervix (Arvanitis and Spandidos, 2008). Increased SIX1expression up-regulates POLA1 and promotes the proliferation and growthof cervical cancer. Moreover, the tumor suppressor microRNA miR-206regulates POLA1 expression in lung cancer cells (Liu et al., 2014b; Cuiet al., 2013).

POLD2 amplification and RPA3 deletion on 7p are correlated with DNAstability and a longer survival of patients of ovarian cancer(Sankaranarayanan et al., 2015). The expression of POLD2 and 6 otherlandscape genes of a network model is associated with the duration ofsurvival for patients with glioblastomas (Bredel et al., 2009).

PPP4C is over-expressed in different cancer types such as colorectal andpancreatic cancer and is further associated with invasion, metastasisand poor prognosis (Weng et al., 2012; Li et al., 2015b). Inhibition ofthe MEK/ERK pathway activates PPP4C as part of PP4 and enhancesNF-kappaB signaling via inactivation of the IKK complex whereasphosphorylated Akt is required for PPP4C-mediated up-regulation of MMP-2and MMP-9 (Brechmann et al., 2012; Li et al., 2015b; Yeh et al., 2004).

PTRH2 is de-regulated in different cancer types includingdown-regulation in lung and breast cancer associated with metastasis andup-regulation in esophageal squamous cell carcinomas and ovarianadenocarcinomas associated with tumor progression (Karmali et al., 2011;Kim et al., 1998; Hua et al., 2013; Fan et al., 2014; Yao et al., 2014).Down-regulation of PTRH2 mediated by E2 binding to ERalpha isaccomplished mainly through the PI3K/Akt pathway and leads to ananti-anoikis effect (Zheng et al., 2014a).

Plk1 phosphorylates RACGAP1 during cytokinesis to create a binding sitefor Ect2 leading to an activation of the Rho/ERK signaling axis whichcan promote metastasis (Kim et al., 2014; Chen et al., 2015b). RACGAP1is over-expressed in different cancer entities including colorectal,breast, liver and lung cancer. RACGAP1 is further associated withprogression and poor prognosis in breast and hepatocellular carcinoma(Wang et al., 2011; Pliarchopoulou et al., 2013; Liang et al., 2013;Imaoka et al., 2015).

Four variants of RACGAP1 P were detectable in 16 families denselyaffected by colorectal cancer (DeRycke et al., 2013).

RGS12 is de-regulated in different cancer types. A single nucleotidepolymorphism (SNP) in the RGS12 gene is associated with overall survivalin late-stage non-small lung cancer and a nonsense-mediated decay(NMD)-resistant frameshift mutation is further associated withmicrosatellite instability (MSI) high colorectal cancer (Williams etal., 2010; Dai et al., 2011; Potocnik et al., 2003). Gbetagamma isacting via PI3-kinase gamma and cSrc to activate the tyrosinephosphorylation of Galphail/2/3 and subsequent association with RGS12resulting in a rapid deactivation of Gaplphai (Huang et al., 2014).

Single nucleotide polymorphisms in RTEL1 are associated with a risk forbrain tumors, such as gliomas. The RTEL1 locus is frequently amplifiedin multiple human cancers including hepatocellular carcinomas andgastrointestinal tract tumors (Wu et al., 2012; Zhao et al., 2014b; Adelet al., 2015). RTEL1 antagonizes homologous recombination by promotingthe disassembly of D loop recombination intermediates in anATP-dependent reaction to eliminate inappropriate recombination eventsand thereby ensures maintenance of genomic integrity (Barber et al.,2008).

SENP1 expression is up-regulated in different cancer types includingprostate, liver and lung cancer and is further associated with cancerprogression and cell proliferation (Bawa-Khalfe et al., 2010; Wang etal., 2013a; Burdelski et al., 2015; Zhang et al., 2016). HGF/c-Met andIL-6 induce SENP1 leading to activation of NFkappaB signaling andepithelial-to-mesenchymal transition (EMT) subsequently resulting incell proliferation and migration (Xu et al., 2015; Zhang et al., 2016).

SETDB2 is frequently deleted or down-regulated in different cancer typesincluding deletions in breast cancer associated with poor prognosis,frameshift mutations in colorectal cancer and deletions of thechromosomal region in chronic lymphocytic leukemia associated withcancer progression (Mabuchi et al., 2001; Parker et al., 2011; Choi etal., 2014; Liu et al., 2015b).

SIPA1L3 is differentially expressed between normal breast epithelium andductal carcinoma in situ and may be associated with regulation of cellproliferation (Abba et al., 2004).

An NHE (Na—H exchange) inhibition suppresses the proliferation ofgastric cancer cells via up-regulation of p21 through a SLC4A8-inducedreduction of chloride concentration without changes in pH (Hosogi etal., 2012).

The biotin uptake of SLC5A6 in cancer cells, such as human breast cancercells, is higher than in normal cells possibly for maintaining the highproliferative status and thus provides a chance for an anti-cancer drugdelivery system (Vadlapudi et al., 2013).

SLCO2A1 is de-regulated in different cancer types including adown-regulation in head and neck squamous cell carcinomas and colorectalcancer and an over-expression in pancreatic and liver cancer (Wlcek etal., 2011; Zolk et al., 2013; Hays et al., 2013; Shang et al., 2015).SLCO2A1 as part of the Cox-2/PGE2 pathway affects especially the PGE2secretion which plays a role in tumorigenesis of colorectal cancercells. SLCO2A1 further mediates the invasion and apoptosis of lungcancer cells via the PI3K/Akt/mTOR pathway (Greenhough et al., 2009; Zhuet al., 2015; Kasai et al., 2016).

SLIT1 is hyper-methylated and down-regulated in different cancer typesincluding cervical, brain, gastric, colorectal and hepatocellular cancer(Zheng et al., 2009; Ghoshal et al., 2010; Kim et al., 2016; Dickinsonet al., 2004; Narayan et al., 2006). SLIT1 is a target of the DNAmethyltransferase DNMT3B resulting in promoter hyper-methylation.

SLIT1 is also a target of the WNT/beta-catenin signaling pathway(Ghoshal et al., 2010; Katoh and Katoh, 2005).

SLIT2 is down-regulated in different tumor entities including breast andrenal cancer and it has been described as tumor suppressor. On the otherhand, also over-expression of SLIT2 in different cancer types includingskin and gastric cancer and an association with cancer progression wasfound. Thus, effects of the SLIT2/Robo1 signaling axis on tumor growthand metastasis seem to be dependent on the cellular context (Alvarez etal., 2013; Shi et al., 2013; Ma et al., 2014c; Qi et al., 2014; Yuan etal., 2016; Prasad et al., 2008). Activation of the SLIT2/Robo1 signalingaxis promotes tumorigenesis through activation of Src signaling,down-regulation of E-cadherin and induction of the Wnt/beta-cateninpathway. Moreover, the tumor-suppressive effects seem to be mediated bythe regulation of the beta-catenin and PI3K signaling pathways resultingin an enhanced beta-catenin/E-cadherin-mediated cell-cell adhesion(Zhang et al., 2015c; Prasad et al., 2008).

SPC25, together with CDCA1, KNTC2 and APC24, is over-expressed incolorectal and gastric cancers when compared to normal mucosae (Kanekoet al., 2009).

STAT4 is de-regulated in different cancer types including adown-regulation in hepatocellular carcinomas and lymphomas associatedwith poor prognosis and an over-expression in colorectal and gastriccancer linked with invasion (Zhou et al., 2014b; Litvinov et al., 2014;Wang et al., 2015a; Cheng et al., 2015). The induction of cytotoxic CD4+T cells via up-regulation of the JAK2/STAT4/perforin pathway can inhibittumor cell growth (Zhou et al., 2014a).

STK33 is de-regulated in different cancer types includingover-expression in hypopharyngeal and liver cancer as well as anassociation with cancer progression and hyper-methylation in colorectalcancer (Moon et al., 2014; Yang et al., 2016; Huang et al., 2015b).STK33 can promote cell migration, invasion and epithelial-to-mesenchymaltransition (EMT) via suppressing of p53, caspase-3 and E-cadherin (Huanget al., 2015b; Wang et al., 2015b).

TAT is down-regulated, hyper-methylated and located at a frequentlydeleted region in hepatocellular carcinomas and shows tumor suppressiveability associated with its pro-apoptotic role in amitochondrial-dependent manner by promoting cytochrome-c release andactivating caspase-9 and PARP (Fu et al., 2010).

Some researchers have reported over-expression of TIMELESS protein andmRNA in hepatocellular carcinoma as well as in colorectal cancer,cervical cancer, lung cancer and prostate cancer. On the other hand,another study reported down-regulation of TIMELESS in hepatocellularcarcinomas. In addition, single nucleotide polymorphism in the TIMELESSgene were not associated with risk of prostate cancer but correlatedwith breast cancer risk (Lin et al., 2008b; Fu et al., 2012; Mazzoccoliet al., 2011; Yoshida et al., 2013; Mao et al., 2013; Markt et al.,2015; Elgohary et al., 2015). In lung cancer, elevated levels ofTIMELESS were associated with poor overall survival (Yoshida et al.,2013).

TIPARP is frequently amplified during oral squamous cell carcinomaprogression and the gene lies on a locus associated with susceptibilityfor ovarian cancer (Goode et al., 2010; Cha et al., 2011).

TLX3 expression associated with T-cell acute lymphoblastic leukemia ismediated by genomic rearrangements, but its prognostic relevance isunder discussion (Ballerini et al., 2008; Ma et al., 2014a; Su et al.,2004). Other genetic lesions that are detectable in TLX3 rearrangedT-ALL cases are deletion of WT1 and FBXW7, an U3-ubiquitin ligasemediating the degradation of Notch1, Myc, Jun and Cyclin E (Van et al.,2008). TMEM44 encodes a multi-transmembrane protein enriched in thebottom portion of taste buds and associated with developmentallyimmature taste cells (Moyer et al., 2009).

TNFRSF6B is over-expressed in different cancer types including breast,cervical, bladder, gastric and liver cancer and is further associatedwith tumor progression (Yang et al., 2010; Lin and Hsieh, 2011; Jiang etal., 2014; Zheng et al., 2014b; Jiang et al., 2016).

Down-regulation of TNFRSF6B induces Fas-ligand-mediated apoptosis by theactivation of FADD, caspase-3, -8,-9 and additionally reduces ERK1/2phosphorylation (Zhou et al., 2013c; Zhang et al., 2015e; Hu et al.,2016).

TOP2A was shown to be up-regulated in adenosquamous carcinoma of thepancreas (Borazanci et al., 2015). TOP2A was described as commonlyaltered at both gene copy number and gene expression level in cancercells, and may play a critical role in chromosome instability in humancancers (Chen et al., 2015c). Alongside with other genes, TOP2A wasshown to be genomically and molecularly aberrated in malignantperipheral nerve sheath tumors and exhibits great promise as apersonalized therapeutic target (Yang and Du, 2013). TOP2A was describedas a de-regulated gene in malignant pleural mesothelioma which may alsobe associated with resistance to cisplatin (Melaiu et al., 2012). TOP2Awas described as an oncogene whose amplification is related to asignificant response to anthracycline-based chemotherapy in breastcancer (Zhang and Yu, 2011). TOP2A deletions and amplifications weredescribed to be prevalent in HER-2 amplified and primary breast tumorsand associated with poor prognosis (Pritchard et al., 2008; Jarvinen andLiu, 2003). TOP2A gene copy numbers were also shown to be elevated in amismatch repair-proficient colorectal cancer subgroup compared to amismatch repair-deficient subgroup, which may provide a survivaladvantage selectively in mismatch repair-proficient tumors (Sonderstrupet al., 2015). TOP2A was described as a potential target for welldifferentiated and de-differentiated liposarcoma (Crago and Singer,2011). TOP2A expression in non-small cell lung cancer was shown to beassociated with tumor histological type and inversely correlated with2-year brain metastases free survival (Huang et al., 2015a). TOP2A wasshown to be associated with increased risk of developing brainmetastases in non-small cell lung cancer (Huang et al., 2015a). TOP2Aexpression was shown to be associated with lower overall anddisease-free survival in patients with endometrial cancer who receivedadjuvant taxane-platinum regimens after surgery (Ito et al., 2016).TOP2A was shown to promote prostate cancer aggressiveness by inducingchromosomal rearrangements of genes that contribute to a more invasivephenotype (Schaefer-Klein et al., 2015). TOP2A was shown to be relatedwith the androgen receptor signaling pathway in a way that contributesto prostate cancer progression and confers sensitivity to treatments(Schaefer-Klein et al., 2015).

TUBGCP2 was shown to be up-regulated in a taxol-resistant ovarian cancercell line and was described to be associated with the sensitization ofthe non-small cell lung carcinoma cell line NCI-H1155 to taxol (Huangand Chao, 2015). TUBGCP2 was shown to be up-regulated in glioblastoma,where its over-expression antagonized the inhibitory effect of the CDK5regulatory subunit-associated tumor suppressor protein 3 on DNA damageG2/M checkpoint activity (Draberova et al., 2015).

TXNIP is a tumor suppressor gene which is down-regulated andhyper-methylated in various cancer entities including breast, lung,stomach and colorectal cancer. It is further associated with tumorprogression and prognosis (Zhou et al., 2011; Zhou and Chng, 2013; Le etal., 2006). TXNIP increases the production of ROS and oxidative stressresulting in cell apoptosis and TXNIP cross-talk with many intracellularsignaling pathways including the cellular glucose uptake as well asc-Myc, p53, HER-2 and p38MAPK/ERK (Zhou and Chng, 2013; Suh et al.,2013; Li et al., 2014; Nie et al., 2015; Shen et al., 2015; Hong andHagen, 2015).

UPF1 is part of the nonsense-mediated mRNA decay (NMD) machinery and mayhave a functional role in prostate cancer progression and metastasis(Yang et al., 2013). Further the UPF1 RNA surveillance gene is commonlymutated in pancreatic adenosquamous carcinoma (Liu et al., 2014a).

USP1 is over-expressed in different cancer types including cervical,lung and gastric cancer, melanoma and sarcoma (Williams et al., 2011;Garcia-Santisteban et al., 2013; Jung et al., 2016). PDGF signalingup-regulates USP1 which de-ubiquitinates and thereby stabilizes theinhibitor of DNA-binding (ID) proteins that are essential factors forproliferation and cancer progression. The same pathway is potentiallyalso involved in the control of chromosome duplication (Wrighton, 2011;Mistry et al., 2013; Jung et al., 2016; Rahme et al., 2016).

A de-regulated expression of UTY is associated with different cancertypes including head and neck, prostate and nasal cancer (Dasari et al.,2001; Lau and Zhang, 2000; Sethi et al., 2009; Llorente et al., 2008).UTY catalyzes the de-methylation of H3K27 peptides and may thereby workas transcriptional repressor (Walport et al., 2014).

Over-expression of VCP in different cancer types including breast, lung,liver, prostate and colorectal cancer is associated with tumorprogression and poor prognosis (Valle et al., 2011; Yamamoto et al.,2003; Cui et al., 2015; Yamamoto et al., 2004; Tsujimoto et al., 2004).The Aurora-B and the Akt kinase can phosphorylate VCP which can thendirectly regulate his downstream targets like p53 and NF-kappaB andthereby influence cell proliferation and survival (Valle et al., 2011;He et al., 2015; Vandermoere et al., 2006; Braun and Zischka, 2008).

WDFY3 was shown to be down-regulated in colorectal cancer (Piepoli etal., 2012).

WDR36 is a target of several cancer-related microRNAs in colorectalcancer (Li et al., 2011b). Loos of WDR36 function leads to an activationof the p53 stress-response pathway, up-regulation of mRNA for Bax, p53and CDNK1A and further causes apoptotic cell death (Gallenberger et al.,2011; Skarie and Link, 2008).

WDR7 expression is de-regulated by copy number alterations in gastriccancer and shows an elevated expression in numerous malignant cell lines(Junnila et al., 2010; Sanders et al., 2000).

Genome-wide association studies identified gene polymorphisms in XXYLT1.It has been proposed that these polymorphisms are susceptibility locifor non-small cell lung cancer development (Zhang et al., 2012).

ZFYVE16 is a phosphorylation target of EGF signaling and can interactwith Smad2 to facilitate TGF-beta signaling thereby playing an importantrole in the regulation of cell growth and proliferation of cancer cells(Chen et al., 2007b; Chen et al., 2007a).

SUMO modification potentiates the negative effect of ZNF131 on estrogensignaling and consequently attenuates estrogen-induced cell growth inbreast cancer cells (Oh and Chung, 2012).

Changes in ZNF292 were described as chronic lymphocytic leukemia driveralterations (Puente et al., 2015). ZNF292 was described as atumor-suppressor gene in colorectal cancer (Takeda et al., 2015). ZNF292was described as an immunogenic antigen with clinical relevance in headand neck squamous cell carcinoma (Heubeck et al., 2013).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 126 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 126, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 126. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 126, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides  according to SEQ ID NO: 1, 3, and 8 Position 1 2 3 4 5 6 7 8 9 10 11 12SEQ ID No 1 A M L E E V N Y I Variants L V L L L L A V L A A V A A L A AV V V V L V A T V T T L T A Q V Q Q L Q A SEQ ID No 3 V L A E I D P K QL V Variants I L A M M I M L M A A A I A L A A V V I V L V A T T I T L TA Q Q I Q L Q A SEQ ID No 8 V L I D D S Q S I I F I Variants V L A M V MM L M A A V A A L A A V V V V L V A T V T T L T A Q V Q Q L Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 126.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 126 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 141and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaCl₄, NaI, NaSCN,ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃,CsCl₄, CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl,LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A-1P).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from small cell lung cancersamples (N=19 A*02-positive samples) with the fragmentation patterns ofcorresponding synthetic reference peptides of identical sequences. Sincethe peptides were directly identified as ligands of HLA molecules ofprimary tumors, these results provide direct evidence for the naturalprocessing and presentation of the identified peptides on primary cancertissue obtained from 14 small cell lung cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from small cell lung cancer tissue samples werepurified and HLA-associated peptides were isolated and analyzed by LC-MS(see examples). All TUMAPs contained in the present application wereidentified with this approach on primary small cell lung cancer samplesconfirming their presentation on primary small cell lung cancer.

TUMAPs identified on multiple small cell lung cancer and normal tissueswere quantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 3A and 3B).An additional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably small cell lung cancer that over- orexclusively present the peptides of the invention. These peptides wereshown by mass spectrometry to be naturally presented by HLA molecules onprimary human small cell lung cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy lung cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from small cell lung cancer, but not on normaltissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. small cell lung cancer cells presenting thederived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, an peptide-HLA molecule complex, which is atleast double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens. It is nowknown, although tumor antigens have the potential to be immunogenic,because tumors arise from the individual's own cells only mutatedproteins or proteins with altered translational processing will be seenas foreign by the immune system. Antigens that are upregulated oroverexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T-cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T-cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to the peptides according to the invention can be enhancedby methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRap geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)—Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription systems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters, TCRexpression cassettes of the present description may contain additionalelements that can enhance transgene expression, including a centralpolypurine tract (cPPT), which promotes the nuclear translocation oflentiviral constructs (Follenzi et al., 2000), and the woodchuckhepatitis virus posttranscriptional regulatory element (wPRE), whichincreases the level of transgene expression by increasing RNA stability(Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID NO: 1 to SEQ ID NO: 126, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL (SEQ ID NO: 144), or may be linkedwithout any additional peptide(s) between them. These constructs canalso be used for cancer therapy, and may induce immune responses bothinvolving MHC I and MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention.

Suitable adjuvants include, but are not limited to, 1018 ISS, aluminumsalts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 126,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 126, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 126 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:126 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 126, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 126.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of small cell lung cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 126 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are small cell lung cancer cells orother solid or hematological tumor cells such as non-small cell lungcancer, small cell lung cancer, renal cell cancer, brain cancer, gastriccancer, colorectal cancer, hepatocellular cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, ovarian cancer, urinary bladder cancer, uterine cancer,gallbladder and bile duct cancer and esophageal cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of small cell lung cancer. The present invention also relatesto the use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a small cell lung cancer marker(poly)peptide, delivery of a toxin to a small cell lung cancer cellexpressing a cancer marker gene at an increased level, and/or inhibitingthe activity of a small cell lung cancer marker polypeptide) accordingto the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length small cell lung cancer marker polypeptides orfragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 126polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the small cell lung cancer markerpolypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating small cell lungcancer, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 126, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition, plant viruses may be used (see, for example,Porta et al. (Porta et al., 1994) which describes the development ofcowpea mosaic virus as a high-yielding system for the presentation offoreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 126.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from small cell lungcancer, the medicament of the invention is preferably used to treatsmall cell lung cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of smallcell lung cancer patients with various HLA-A HLA-B and HLA-C alleles. Itmay contain MHC class I and MHC class II peptides or elongated MHC classI peptides. In addition to the tumor associated peptides collected fromseveral small cell lung cancer tissues, the warehouse may containHLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparisonof the magnitude of T-cell immunity induced by TUMAPS in a quantitativemanner and hence allow important conclusion to be drawn on the capacityof the vaccine to elicit anti-tumor responses. Secondly, they functionas important positive control peptides derived from a “non-self” antigenin the case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, small cell lung cancer samplesfrom patients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (smallcell lung cancer) compared with a range of normal organs and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromsmall cell lung cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from small cell lung cancer cells and since it wasdetermined that these peptides are not or at lower levels present innormal tissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for small cell lung cancer. Presence of groups of peptides canenable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A to 1P show the over-presentation of various peptides in normaltissues (white bars) and cancer (black bars). FIGS. 1A to 1E show theover-presentation of various peptides in normal tissues (white bars) andsmall cell lung cancer (black bars). FIG. 1A) CCNE2, Peptide: AMLEEVNYI(SEQ ID NO: 1)—Tissues from left to right: 2 adipose tissues, 3 adrenalglands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7 brains, 5breasts, 2 cartilages, 3 gallbladders, 5 hearts, 14 kidneys, 19 largeintestines, 20 livers, 45 lungs, 4 lymph nodes, 7 nerves, 3 ovaries, 10pancreas, 1 peritoneum, 5 pituitary glands, 6 placentas, 3 pleuras, 3prostates, 7 salivary glands, 5 skeletal muscles, 6 skins, 3 smallintestines, 4 spleens, 5 stomachs, 6 testis, 3 thymi, 3 thyroid glands,9 tracheas, 2 ureters, 6 urinary bladders, 2 uteri, 6 esophagi, 19 SCLCcancer samples. The peptide has additionally been detected on 2/17chronic lymphocytic leukemias, 1/20 pancreatic cancer cell lines, 1/27colorectal cancer, 4/16 Non-Hodgkin lymphomas, 3/19 pancreatic cancers.FIG. 1B) IFT81, Peptide: VLAEIDPKQLV (SEQ ID NO: 3)—Tissues from left toright: 2 adipose tissues, 3 adrenal glands, 4 blood cells, 10 bloodvessels, 6 bone marrows, 7 brains, 5 breasts, 2 cartilages, 3gallbladders, 5 hearts, 14 kidneys, 19 large intestines, 20 livers, 45lungs, 4 lymph nodes, 7 nerves, 3 ovaries, 10 pancreas, 1 peritoneum, 5pituitary glands, 6 placentas, 3 pleuras, 3 prostates, 7 salivaryglands, 5 skeletal muscles, 6 skins, 3 small intestines, 4 spleens, 5stomachs, 6 testis, 3 thymi, 3 thyroid glands, 9 tracheas, 2 ureters, 6urinary bladders, 2 uteri, 6 esophagi, 19 SCLC cancer samples. Thepeptide has additionally been detected on 2/16 liver cancers, 2/20ovarian cancers, 1/20 esophageal cancer. FIG. 1C) POLA1, Peptide:GLDPTQFRV (SEQ ID NO: 39) Tissues from left to right: 2 adipose tissues,3 adrenal glands, 4 blood cells, 10 blood vessels, 6 bone marrows, 7brains, 6 breasts, 2 cartilages, 1 eye, 3 gallbladders, 5 hearts, 14kidneys, 19 large intestines, 20 livers, 45 lungs, 4 lymph nodes, 7nerves, 3 ovaries, 10 pancreases, 1 peritoneum, 5 pituitary glands, 6placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5 skeletalmuscles, 6 skins, 3 small intestines, 4 spleens, 5 stomachs, 6 testis, 3thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinary bladders, 2uteri, 6 esophagi, 19 SCLC cancer samples. The peptide has additionallybeen detected on 1/20 pancreatic cancer cell line, 1/16 Non-Hodgkinlymphoma, 1/20 ovarian cancer, 1/20 urinary bladder cancer. FIG. 1D)LOXL4, Peptide: GLLEVQVEV (SEQ ID NO: 40)—Tissues from left to right: 2adipose tissues, 3 adrenal glands, 4 blood cells, 10 blood vessels, 6bone marrows, 7 brains, 6 breasts, 2 cartilages, 1 eye, 3 gallbladders,5 hearts, 14 kidneys, 19 large intestines, 20 livers, 45 lungs, 4 lymphnodes, 7 nerves, 3 ovaries, 10 pancreases, 1 peritoneum, 5 pituitaryglands, 6 placentas, 3 pleuras, 3 prostates, 7 salivary glands, 5skeletal muscles, 6 skins, 3 small intestines, 4 spleens, 5 stomachs, 6testis, 3 thymi, 3 thyroid glands, 9 tracheas, 3 ureters, 6 urinarybladders, 2 uteri, 6 esophagi, 19 SCLC cancer samples. The peptide hasadditionally been detected on 1/20 pancreatic cancer cell line, 1/20ovarian cancer. FIG. 1E) USP1, Peptide: SLQSLIISV (SEQ ID NO: 5)—Samplesfrom left to right: 3 cell-lines (1 blood, 1 pancreatic), 2 normaltissues (1 lymph node, 1 spleen), 24 cancer tissues (2 leukocyticleukemia cancers, 1 breast cancer, 1 gallbladder cancer, 5 lung cancers,6 lymph node cancers, 2 ovarian cancers, 2 prostate cancers, 2 skincancers, 1 urinary bladder cancer, 2 uterine cancers). FIGS. 1F to Pshow the over-presentation of various peptides in normal tissues (whitebars) and cancer (black bars). FIG. 1F) Gene Symbol: GPR98, Peptide:GLLGDIAIHL (SEQ ID NO.: 7)—Tissues from left to right: 1 cell line(blood cells), 2 normal tissues (1 brain, 1 pituitary gland), 35 cancertissues (27 brain cancers, 1 breast cancer, 1 liver cancer, 4 lungcancers, 2 uterus cancers). FIG. 1G) Gene Symbol: ITPR1, Peptide:ILIETKLVL (SEQ ID NO.: 14)—Tissues from left to right: 2 cell lines (1prostate, 1 skin), 2 normal tissues (1 lymph node, 1 spleen), 26 cancertissues (11 leukocytic leukemia cancers, 1 kidney cancer, 6 lungcancers, 4 lymph node cancers, 1 ovarian cancer, 1 prostate cancer, 1stomach cancer, 1 testis cancer). FIG. 1H) Gene Symbol: ATP2C1, Peptide:GLYSKTSQSV (SEQ ID NO.: 33)—Tissues from left to right: 2 cell lines (1blood cells, 1 pancreas), 4 normal tissues (1 adrenal gland, 1 colon, 2lungs), 41 cancer tissues (1 leukocytic leukemia cancer, 2 breastcancers, 3 esophageal cancers, 5 head-and-neck cancers, 1 colon cancer,1 liver cancer, 14 lung cancers, 3 lymph node cancers, 5 ovariancancers, 1 prostate cancer, 3 urinary bladder cancers, 2 uteruscancers). FIG. 1I) Gene Symbol: NEDD1, Peptide: SLSGEIILHSV (SEQ ID NO.:45)—Tissues from left to right: 3 cell lines (2 pancreases, 1 skin), 14cancer tissues (2 breast cancers, 3 head-and-neck cancers, 1 coloncancer, 5 lung cancers, 1 skin cancer, 2 urinary bladder cancers). FIG.1J) Gene Symbol: SLC4A8, Peptide: VLLSGLTEV (SEQ ID NO.: 59)—Tissuesfrom left to right: 8 cancer tissues (1 leukocytic leukemia cancer, 4brain cancers, 2 lung cancers, 1 rectum cancer). FIG. 1K) Gene Symbol:ECT2, Peptide: KAIGSLKEV (SEQ ID NO.: 72)—Tissues from left to right: 1cell line (1 pancreas), 12 cancer tissues (1 esophageal cancer, 1 coloncancer, 1 rectum cancer, 5 lung cancers, 2 ovarian cancers, 1 stomachcancer, 1 uterus cancer). FIG. 1L) Gene Symbol: XXYLT1, Peptide:RLLEPAQVQQL (SEQ ID NO.: 79)—Tissues from left to right: 4 cell lines (1blood cells, 2 pancreases, 1 skin), 2 normal tissues (1 colon, 1spleen), 29 cancer tissues (2 brain cancers, 1 breast cancer, 2esophageal cancers, 1 head-and-neck cancer, 1 rectum cancer, 1 livercancer, 8 lung cancers, 4 lymph node cancers, 4 ovarian cancers, 1 skincancer, 2 urinary bladder cancers, 2 uterus cancers). FIG. 1M) GeneSymbol: TSEN34, Peptide: LLAEIGAVTLV (SEQ ID NO.: 81)—Tissues from leftto right: 8 cell lines (5 blood cells, 1 pancreas, 2 skin), 30 cancertissues (1 bile duct cancer, 1 myeloid cells cancer, 1 leukocyticleukemia cancer, 2 breast cancers, 2 head-and-neck cancers, 1 coloncancer, 2 liver cancers, 11 lung cancers, 1 lymph node cancer, 3 ovariancancers, 1 prostate cancer, 2 skin cancers, 2 urinary bladder cancers).FIG. 1N) Gene Symbol: MCM2, Peptide: FLPEAPAEL (SEQ ID NO.: 111)—Tissuesfrom left to right: 5 cell lines (5 blood cells), 19 cancer tissues (5leukocytic leukemia cancers, 1 myeloid cells cancer, 1 bone marrowcancer, 1 gallbladder cancer, 1 lung cancer, 8 lymph node cancers, 1stomach cancer, 1 uterus cancer). FIG. 1O) Gene Symbol: LIG3, Peptide:PGVIKTV (SEQ ID NO.: 112)—Tissues from left to right: 1 cell line (1blood cells), 10 cancer tissues (1 leukocytic leukemia cancer, 1 coloncancer, 4 lung cancers, 3 ovarian cancers, 1 urinary bladder cancer).FIG. 1P) Gene Symbol: STK33, Peptide: SLIDDNNEINL (SEQ ID NO.:119)—Tissues from left to right: 3 cell lines (3 blood cells), 1 normaltissue (1 trachea), 13 cancer tissues (3 brain cancers, 2 lung cancers,2 lymph node cancers, 3 ovarian cancers, 1 prostate cancer, 2 uteruscancers).

FIGS. 2A to 2D show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in small cell lung cancer in a panel of normal tissues (whitebars) and 10 small cell lung cancer samples (black bars). FIG. 2A.Genesymbol: MEX3A; FIG. 2B Genesymbol: ECT2; FIG. 2C: Genesymbol: CCNE2;FIG. 2D: Genesymbol: TIMELESS.

FIGS. 3A-3B shows exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining. (FIG. 3A): Gene symbol: SLIT1,SLIT2, Peptide: SLYDNQITTV (SEQ ID NO: 130); (FIG. 3B): Gene symbol:TLX3, Peptide: SLAPAGVIRV (SEQ ID NO: 128).

FIGS. 4A-4C shows exemplary results of peptide-specific in vitro CD8+ Tcell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complexwith SeqID No 80 peptide ILMDPSPEYA (FIG. 4A, left panel), SeqID No 82peptide ALSSVIKEL (FIG. 4B, left panel) and SeqID No 110 peptideGLTETGLYRI (FIG. 4C, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*02/SeqID No 80 (FIG. 4A), A*02/SeqID No 82(FIG. 4B) or A*02/SeqID No 110 (FIG. 4C). Right panels (FIGS. 4A, 4B and4C) show control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, Mich.,USA & Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA) Tissue Solutions Ltd(Glasgow, UK).

Normal tissues were obtained from Asterand (Detroit, Mich., USA &Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); UniversityHospital Tübingen (Tübingen, Germany)

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose small cell lung cancer samples to a baseline ofnormal tissue samples. Presentation profiles of exemplary over-presentedpeptides are shown in FIGS. 1A-1P. Presentation scores for exemplarypeptides are shown in Table 8.

TABLE 8 Presentation scores. The table lists peptidesthat are very highly over-presented on tumorscompared to a panel of normal tissues (+++),highly over-presented on tumors compared to apanel of normal tissues (++) or over-presentedon tumors compared to a panel of normal tis-sues (+). The panel of normal tissues co-nsidered relevant for comparison with tumorsconsisted of: adipose tissue, adrenal gland,blood cells, blood vessel, bone marrow, brain,esophagus, eye, gallbladder, heart, kidney,large intestine, liver, lung, lymph node,nerve, pancreas, parathyroid gland, perito-neum, pituitary, pleura, salivary gland,skeletal muscle, skin, small intestine,spleen, stomach, thymus, thyroid gland,trachea, ureter, urinary bladder. SEQ ID Peptide NO: SequencePresentation   1 AMLEEVNYI +++   2 VMFNFPDQATV ++   3 VLAEIDPKQLV +++  4 GLLDPGMLVNI +   5 SLQSLIISV +   6 SIMDYVVFV ++   7 GLLGDIAIHL +++  8 VLIDDSQSIIFI +++   9 AAAPGEALHTA +  10 ILAAGFDGM +  11 KLFAIPILL +++ 15 SLLTAISEV +  16 VILDLPLVI +++  17 SLMLVTVEL +  19 VLLTTAVEV +  20MLDEILLQL +  23 YQIDTVINL +  24 FLMEEVHMI +  26 KMLDEAVFQV +  27SLDIITITV +++  29 NLISQLTTV ++  31 RLLQDPVGV +  35 FMGDVFINV +  37SLFYNELHYV +  39 GLDPTQFRV +++  40 GLLEVQVEV +++  41 KAYQELLATV +++  42GLLEDERALQL +++  43 YLWSEVFSM +++  44 ALIVGIPSV +++  45 SLSGEIILHSV +++ 46 ALWVAVPKA +++  47 GLLEALLKI +++  49 RLALNTPKV ++  50 FLLSQIVAL +++ 51 ILDEAGVKYFL +++  52 ILASFMLTGV +++  54 HLFDIILTSV ++  55 LLIADNPQL+++  56 SLFSQMGSQYEL +++  57 VLIGDVLVAV +++  58 VLLNINGIDL +++  59VLLSGLTEV +++  60 VVSGATETL +++  61 YQAPYFLTV +++  62 VMLPIGAVVMV +++ 63 LLMSTENEL ++  64 VLFHQLQEI +  65 VMYDLITEL ++  66 YLNLISTSV +++  67MLYDIVPVV +  68 FLFPVYPLI +  69 KLFDRSVDL ++  70 TLLWKLVEV +++  72KAIGSLKEV +++  73 SLSSYTPDV +++  74 FLDSLSPSV +++  75 SLDLHVPSL +++  76VLTTVMITV +++  78 RIIDPEDLKALL +++  79 RLLEPAQVQQL ++  80 ILMDPSPEYA +++ 81 LLAEIGAVTLV ++  82 ALSSVIKEL +  83 KLLEIDIDGV +  84 KMFENEFLL +  85FAYDGKDYLTL +  86 KVIDYVPGI +  87 LLQNNLPAV +  88 TLHRETFYL +++  89IQHDLIFSL +  90 TLVDNISTMAL +  95 ALYSKGILL +  96 NLLKLIAEV +  97ALLDGTVFEI ++  98 ALVDHLNVGV +  99 QMLEAIKALEV ++ 100 VADPETRTV + 101AMNSQILEV + 103 SLLEYQMLV + 105 SMYDKVLML + 106 KMPDDVWLV + 107AMYGTKLETI + 110 GLTETGLYRI +++ 111 FLPEAPAEL +++ 112 LLLPGVIKTV +++ 114ALLEPGGVLTI +++ 115 ALLPSDCLQEA +++ 116 ALLVRLQEV +++ 117 FLLDSAPLNV +118 KLPSFLANV +++ 119 SLIDDNNEINL + 120 SLAADIPRL ++ 121 YMLEHVITL + 124SLITDLQTI ++ 125 LLSEPSLLRTV +++ 126 AAASLIRLV +++

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd(Glasgow, UK)

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples,Italy); ProteoGenex Inc. (Culver City, Calif., USA); University HospitalHeidelberg (Heidelberg, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values. Exemplary expression profilesof source genes of the present invention that are highly over-expressedor exclusively expressed in small cell lung cancer are shown in FIGS.2A-2D. Expression scores for further exemplary genes are shown in Table9.

TABLE 9 Expression scores. The table lists peptidesfrom genes that are very highly over-expressed in tumors compared to a panel ofnormal tissues (+++), highly over-expressedin tumors compared to a panel of normaltissues (++) or over-expressed in tumorscompared to a panel of normal tissues (+).The baseline for this score was calculatedfrom measurements of the following relevantnormal tissues: adipose tissue, adrenalgland, artery, blood cells, bone marrow,brain, cartilage, colon, esophagus, gall-bladder, heart, kidney, liver, lung, lymphnode, pancreas, pituitary, rectum, salivarygland, skeletal muscle, skin, small intestine,spleen, stomach, thymus, thyroid gland,trachea, urinary bladder, vein. In caseexpression data for several samples of thesame tissue type were available, the arith-metic mean of all respective samples was used for the calculation. SEQID Gene No Sequence Expression   1 AMLEEVNYI +++  10 ILAAGFDGM ++  25GLSETILAV ++  39 GLDPTQFRV ++  41 KAYQELLATV +++  42 GLLEDERALQL +++  51ILDEAGVKYFL +  70 TLLWKLVEV +++  72 KAIGSLKEV +++  83 KLLEIDIDGV ++  88TLHRETFYL +++  91 KLQDGVHII +  92 YLQDYTDRV +++  95 ALYSKGILL ++  96NLLKLIAEV ++ 110 GLTETGLYRI + 111 FLPEAPAEL +++ 121 YMLEHVITL ++ 122SMMPDELLTSL + 123 KLDKNPNQV +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 142) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO:143), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specific multimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Small Cell Lung Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A and 3B together withcorresponding negative controls. Results for 4 peptides from theinvention are summarized in Table 10.

TABLE 10A in vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immuno-genicity experiments conducted by the applicant for the peptides of theinvention. <20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++ Seq IDSequence wells 128 SLAPAGVIRV + 129 RVADYIVKV + 130 SLYDNQITTV ++ 132NLLAEIHGV +

TABLE 10B in vitro immunogenicity of HLA class Ipeptides of the invention Exemplary results of in vitro immuno-genicity experiments conducted by the applicant for HLA-A*02 restrictedpeptides of the invention. Results ofin vitro immunogenicity experiments areindicated. Percentage of positive wellsand donors (among evaluable) are sum- marized as indicated <20% =+; 20%-49% = ++; 50%-69% = +++; >=70% = ++++ Seq Wells ID positive NoSequence [%]   2 VMFNFPDQATV +   4 GLLDPGMLVNI ++   6 SIMDYVVFV +  11KLFAIPILL ++++  39 GLDPTQFRV ++++  80 ILMDPSPEYA +++  82 ALSSVIKEL + 110GLTETGLYRI +++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores.Binding of HLA-class I restricted peptidesto HLA-A*02:01 was ranged by peptide exchange yield: ≥10% = +; ≥20% =++; ≥50 = +++; ≥75% = ++++ Seq ID Peptide No Sequence exchange   1AMLEEVNYI +++   2 VMFNFPDQATV +++   3 VLAEIDPKQLV +++   4 GLLDPGMLVNI+++   5 SLQSLIISV ++++   6 SIMDYVVFV ++++   7 GLLGDIAIHL +++   9AAAPGEALHTA ++  11 KLFAIPILL +++  12 MLFEGLDLVSA +++  13 FLTAFLVQI +++ 14 ILIETKLVL +++  15 SLLTAISEV +++  16 VILDLPLVI +++  17 SLMLVTVEL +++ 18 ALGEISVSV +++  19 VLLTTAVEV +++  20 MLDEILLQL +++  21 TMEEMIFEV +++ 22 LLPEKSWEI +++  23 YQIDTVINL +++  24 FLMEEVHMI +++  25 GLSETILAV ++++ 26 KMLDEAVFQV ++++  27 SLDIITITV ++++  28 ILVSQLEQL ++++  30 KMLGLTVSL+++  31 RLLQDPVGV ++++  32 ALTSLELEL +++  34 LVFEGIMEV +++  35 FMGDVFINV++++  36 RMDGAVTSV +++  37 SLFYNELHYV +++  38 GLISSLNEI ++++  39GLDPTQFRV +++  40 GLLEVQVEV +++  41 KAYQELLATV +++  42 GLLEDERALQL +++ 43 YLWSEVFSM +++  44 ALIVGIPSV ++++  45 SLSGEIILHSV ++  46 ALWVAVPKA+++  47 GLLEALLKI +++  48 SLIGLDLSSV +++  49 RLALNTPKV ++  50 FLLSQIVAL+++  51 ILDEAGVKYFL +++  52 ILASFMLTGV ++++  53 LLSEEHITL ++++  54HLFDIILTSV ++++  55 LLIADNPQL ++++  56 SLFSQMGSQYEL +++  57 VLIGDVLVAV++++  58 VLLNINGIDL ++++  59 VLLSGLTEV ++++  60 VVSGATETL ++++  61YQAPYFLTV +++  62 VMLPIGAVVMV +++  63 LLMSTENEL +++  64 VLFHQLQEI +++ 65 VMYDLITEL +++  66 YLNLISTSV +++  67 MLYDIVPVV +++  68 FLFPVYPLI +++ 69 KLFDRSVDL ++  70 TLLWKLVEV ++  71 FIFEQVQNV +++  72 KAIGSLKEV +++ 73 SLSSYTPDV +++  74 FLDSLSPSV +++  75 SLDLHVPSL +++  77 AIIDGKIFCV +++ 78 RIIDPEDLKALL ++++  79 RLLEPAQVQQL +++  80 ILMDPSPEYA +++  81LLAEIGAVTLV +++  82 ALSSVIKEL +++  83 KLLEIDIDGV +++  84 KMFENEFLL +++ 85 FAYDGKDYLTL +++  86 KVIDYVPGI +++  87 LLQNNLPAV ++++  88 TLHRETFYL++++  89 IQHDLIFSL +++  90 TLVDNISTMAL +++  91 KLQDGVHII +++  92YLQDYTDRV +++  93 ALRETVVEV +++  94 ALFPVAEDISL +++  95 ALYSKGILL ++++ 96 NLLKLIAEV ++++  97 ALLDGTVFEI +++  98 ALVDHLNVGV +++  99 QMLEAIKALEV++++ 100 VADPETRTV + 101 AMNSQILEV +++ 102 ALFARPDLLLL ++++ 103SLLEYQMLV ++++ 104 TLIQFTVKL +++ 105 SMYDKVLML ++++ 106 KMPDDVWLV ++++107 AMYGTKLETI ++++ 108 ILLDDQFQPKL ++++ 109 SLFERLVVL ++++ 110GLTETGLYRI +++ 111 FLPEAPAEL +++ 112 LLLPGVIKTV +++ 114 ALLEPGGVLTI +++115 ALLPSDCLQEA +++ 116 ALLVRLQEV ++++ 117 FLLDSAPLNV ++++ 118 KLPSFLANV+++ 120 SLAADIPRL +++ 121 YMLEHVITL +++ 122 SMMPDELLTSL +++ 123KLDKNPNQV +++ 124 SLITDLQTI +++ 125 LLSEPSLLRTV +++ 126 AAASLIRLV +

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The invention claimed is:
 1. A method of eliciting an immune response ina patient who has cancer, comprising administering to said patient apopulation of activated T cells that selectively recognize cells thataberrantly present a peptide consisting of the amino acid sequence ofSEQ ID NO: 42, wherein said cancer is selected from the group consistingof lung cancer, melanoma, liver cancer, breast cancer, uterine cancer,Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile ductcancer, colon or rectum cancer, urinary bladder cancer, kidney cancer,leukemia, ovarian cancer, esophageal cancer, brain cancer, gastriccancer, and prostate cancer.
 2. The method of claim 1, wherein the Tcells are autologous to the patient.
 3. The method of claim 1, whereinthe T cells are obtained from a healthy donor.
 4. The method of claim 1,wherein the T cells are obtained from tumor infiltrating lymphocytes orperipheral blood mononuclear cells.
 5. The method of claim 1, whereinthe activated T cells are expanded in vitro.
 6. The method of claim 1,wherein the population of activated T cells are administered in the formof a composition.
 7. The method of claim 6, wherein the compositionfurther comprises an adjuvant.
 8. The method of claim 7, wherein theadjuvant is selected from the group consisting of anti-CD40 antibody,imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. The methodof claim 1, wherein the activated T cells are cytotoxic T cells producedby contacting T cells with an antigen presenting cell that presents thepeptide in a complex with an MEW class I molecule on the surface of theantigen presenting cell, for a period of time sufficient to activatesaid T cell.
 10. The method of claim 9, wherein the contacting is invitro.
 11. The method of claim 1, wherein the cancer is lung cancer. 12.The method of claim 1, wherein the cancer is gastric cancer.
 13. Themethod of claim 1, wherein the cancer is esophageal cancer.
 14. Themethod of claim 1, wherein the cancer is brain cancer.
 15. The method ofclaim 1, wherein the activated T cells release a cytokine.
 16. Themethod of claim 1, wherein the immune response is capable of killingcancer cells that present a peptide consisting of the amino acidsequence of SEQ ID NO:
 42. 17. A method of eliciting an immune responsein a patient who has lung cancer, melanoma, liver cancer, breast cancer,uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladdercancer, bile duct cancer, colon or rectum cancer, urinary bladdercancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer,brain cancer, gastric cancer, or prostate cancer, comprisingadministering to said patient a composition comprising a peptide in theform of a pharmaceutically acceptable salt and an adjuvant, wherein saidpeptide consists of the amino acid sequence of SEQ ID NO: 42, therebyinducing a T-cell response to the lung cancer, melanoma, liver cancer,breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer,gallbladder cancer, bile duct cancer, colon or rectum cancer, urinarybladder cancer, kidney cancer, leukemia, ovarian cancer, esophagealcancer, brain cancer, gastric cancer, or prostate cancer.
 18. The methodof claim 17, wherein the T cell response is a cytotoxic T cell response.19. The method of claim 17, wherein the adjuvant is selected from thegroup consisting of anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 20. The method of claim 17,wherein the immune response is capable of killing cancer cells thatpresent a peptide consisting of the amino acid sequence of SEQ ID NO:42.